Transcription factor genes and proteins from helianthus annuus, and transgenic plants including the same

ABSTRACT

A polynucleotide having at least 80% sequence identity with the full-length nucleotide sequence of SEQ ID NO: 1 and substantially identical polynucleotides; an isolated polypeptide having at least 80% sequence identity with the full-length amino acid sequence of SEQ ID NO: 2 and substantially identical polypeptides; and polynucleotides encoding the HaWRKY76 polypeptide and substantially identical polypeptides are described. Also described are vectors and recombinant expression cassettes containing the cDNA polynucleotide, a polynucleotide encoding the HaWRKY76 polypeptide, or substantially identical polynucleotides. Transgenic plants containing such expression cassettes, related methods and uses are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/307,333, filed Oct. 27, 2016, which is a National Phase applicationclaiming priority to PCT/GB2015/051269 filed Apr. 30, 2015, which claimspriority to 61/986,730, filed Apr. 30, 2014, the entire contents ofwhich are hereby expressly incorporated by reference in its entiretyincluding, without limitation, the specification, claims, and abstract,as well as any figures, tables, or drawings thereof.

BACKGROUND

Abiotic environmental stresses, such as drought, salinity, wind, heat,and cold, are major limiting factors of plant growth and crop yield.Prolonged or continuous exposure to drought conditions causes majoralterations in the plant metabolism that ultimately lead to cell deathand, consequently, losses in crop yield. High salt content in some soilsresults in less water being available for cell intake; thus, high saltconcentration has an effect on plants similar to the effect of droughton plants. Under freezing temperatures, plant cells lose water as aresult of ice formation within the plant. Because crop damage fromabiotic stresses is predominantly due to dehydration, water availabilityis an important aspect of the abiotic stresses and their effects onplant growth. Losses in crop yield of major crops caused by thesestresses represent a major economic factor and contribute to foodshortages in many underdeveloped countries.

Most plants have evolved protective mechanisms against dehydrationcaused by abiotic stress. However, if the severity and duration of theabiotic stress conditions are too great, the effects on development,growth, and yield of most crop plants are profound. Developing plantsefficient in water use is therefore a strategy that has the potential tobenefit human life. Many agricultural companies have attempted toidentify genes that could confer tolerance to abiotic stress responses,in an effort to develop transgenic abiotic stress-tolerant crop plants.For example, the genome of the plant model Arabidopsis was the first tobe sequenced and released by 2000. Although some genes that play a rolein stress responses or efficient water utilization in plants have beencharacterized, the characterization and cloning of plant genes thatconfer the desired stress tolerance and/or efficient water utilizationcharacteristics remain largely fragmented and incomplete.

The sunflower belongs to the Asteraceae family, whose members represent10% of the flowering plants. However, the genome sequence of thisspecies is largely unknown and the huge quantity of expressed sequencetags (ESTs) from Helianthus (sunflower) species available in publicdatabases have not yet been well explored.

SUMMARY

Embodiments relate to a polynucleotide having at least 85% sequenceidentity with the full-length nucleotide sequence of SEQ ID NO: 1 or SEQID NO: 9, or substantially identical variant polynucleotides, forexample as shown in SEQ ID NO:11. A polynucleotide according to specificembodiments contains at least one of: (a) adenine as the nucleotidecorresponding to position 817 of the full-length nucleotide sequence ofSEQ ID NO: 1 or 11; and (b) cytosine as the nucleotide corresponding toposition 808 of the full-length nucleotide sequence of SEQ ID NO: 1 or11. In other embodiments, the polynucleotide may also contain at leastone of the following: (c) cytosine as the nucleotide corresponding toposition 33 of the full-length nucleotide sequence of SEQ ID NO: 1 or11; (d) cytosine as the nucleotide corresponding to position 259 of thefull-length nucleotide sequence of SEQ ID NO: 1 or 11; and (e) guanineas the nucleotide corresponding to position 315 of the full-lengthnucleotide sequence of SEQ ID NO: 1 or 11.

Embodiments also relate to vectors comprising a polynucleotide asdescribed herein, recombinant expression cassettes comprising apolynucleotide described herein operably linked to a promoter,transgenic plants comprising such a recombinant expression cassette, andmethods for producing such vectors, cassettes, and transgenic plants.

Embodiments also relate to isolated polypeptides having at least 85%sequence identity with the full-length amino acid sequence of SEQ ID NO:2 or 12, or substantially identical polypeptides, for example as shownin SEQ ID NO:12. Polypeptides according to specific embodiments containat least one of: (a) proline as the amino acid corresponding to position270 of the full-length amino acid sequence of SEQ ID NO: 2 or 12; and(b) proline as the amino acid corresponding to position 87 of thefull-length amino acid sequence of SEQ ID NO: 2 or 12. In embodiments, apolypeptide disclosed herein may contain at least one of: (c) proline asthe amino acid corresponding to position 260 of the full-length aminoacid sequence of SEQ ID NO: 2 or 12; (d) serine as the amino acidcorresponding to position 123 of the full-length amino acid sequence ofSEQ ID NO: 2 or 12; (e) leucine as the amino acid corresponding toposition 14 of the full-length amino acid sequence of SEQ ID NO: 2 or12; (f) leucine as the amino acid corresponding to position 22 of thefull-length amino acid sequence of SEQ ID NO: 2 or 12; and (g) serine asthe amino acid corresponding to position 23 of the full-length aminoacid sequence of SEQ ID NO: 2 or 12.

Some embodiments relate to polynucleotides that encode a polypeptidedescribed herein. A vector comprising a polynucleotide that encodes apolypeptide described herein, a recombinant expression cassettecomprising such a polynucleotide operably linked to a promoter,transgenic plants comprising such a recombinant expression cassette, andmethods for producing such products are also provided.

Some embodiments relate to recombinant expression cassettes comprisingan isolated polynucleotide operably linked to a promoter, wherein thepolynucleotide is a member selected from the group consisting of: (a) apolynucleotide that encodes the polypeptide of SEQ ID NO: 2 or a variantthereof, for example SEQ ID NO: 2; and (b) the polynucleotide of SEQ IDNO: 1 or 9 or a variant thereof.

The invention also relates to transgenic plants comprise such arecombinant expression cassette. The invention further provides a methodof producing a transgenic plant comprising: (a) introducing into a plantcell such a recombinant expression cassette; and (b) culturing the plantcell under plant growing conditions to produce the transgenic plant. Theinvention also provides methods for modulating a plant phenotype, forexample increasing yield of a plant under non-stress conditionscomprising introducing and expressing a polynucleotide described herein,for example the polynucleotide of SEQ ID NO: 1 or a variant thereof, forexample SEQ ID NO: 11, or SEQ ID NO: 9 or a variant thereof. Also, theinvention provides a method for increasing stress tolerance of a plantto severe and/or moderate stress comprising introducing and expressing apolynucleotide described herein, for example the polynucleotide of SEQID NO: 1 or a variant thereof, for example SEQ ID NO: 11, or SEQ ID NO:9 or a variant thereof.

In another aspect, the invention provides the use of a polynucleotidedescribed herein, for example the polynucleotide of SEQ ID NO: 1 or avariant thereof, for example SEQ ID NO: 11, or SEQ ID NO: 9 or a variantthereof or a polypeptide encoded by any of these polynucleotides inaltering a plant phenotype, specifically in increasing yield of a plantunder non-stress conditions and/or for increasing stress tolerance of aplant to severe and moderate stress.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is an isolated nucleotide sequence (cDNA) of the HaWRKY76polynucleotide.

SEQ ID NO: 2 is an isolated amino acid sequence of the HaWRKY76polypeptide.

SEQ ID NO: 3 is an isolated nucleotide sequence of the HaT131007971polynucleotide (as identified in the Helia database).

SEQ ID NO: 4 is an isolated amino acid sequence of the HaT131007971polypeptide (as identified in the Helianthus annuus cv. XRQtranscriptome portal).

SEQ ID NO: 5 is an isolated nucleotide sequence of the HuCL13748C001polynucleotide (as identified in the Helia database).

SEQ ID NO: 6 is an isolated amino acid sequence of the HuCL13748C001polypeptide (as identified in the Helia database).

SEQ ID NO: 7 is a conserved motif

SEQ ID NO: 8 is a conserved motif

SEQ ID NO: 9 is the HaWRKY76 genomic DNA.

SEQ ID NO: 10 is the promoter DNA.

SEQ ID NO: 11 is an isolated variant nucleotide sequence (cDNA) of theHaWRKY76 polynucleotide

SEQ ID NO: 12 is an isolated amino acid sequence of the HaWRKY76polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison of the amino acid sequence of the HaT131007971polypeptide of SEQ ID NO: 4 with the amino acid sequence of the HaWRKY76polypeptide of SEQ ID NO: 2.

FIG. 2 is a comparison of the nucleotide sequence of the HaT131007971polynucleotide of SEQ ID NO: 3 with the nucleotide sequence of theHaWRKY76 polynucleotide of SEQ ID NO: 1.

FIG. 3 is a comparison of the amino acid sequence of the HuCL13748C001polypeptide of SEQ ID NO: 6 with the amino acid sequence of the HaWRKY76polypeptide of SEQ ID NO: 2.

FIG. 4 shows the nucleotide sequence of the HaWRKY76 polynucleotide ofSEQ ID NO: 1 with a related sequence.

FIG. 5 shows the HaWRKY76 expression levels in the roots, hypocotyls,and cotyledons in 5-day-old sunflower seedlings. Different lettersindicate samples that are significantly different (p value<0.05). Anillustrative photograph of a sunflower plant in the developmental stageused to take the RNA samples is shown in the right side.

FIG. 6A shows the Ha WRKY76 expression levels in 5-day-old sunflowerseedlings, in standard conditions (left) and under severe drought stress(right) and FIG. 6B shows the expression levels of sunflower R2 plantssubjected to a continuous and severe drought stress during fifteen days.Different letters indicate samples that are significantly different (pvalue<0.05). FIG. 6C is an illustrative photograph of sunflower plantssubjected to severe water stress and used to take the RNA samples.

FIG. 7 shows the Ha WRKY76 expression levels of three homozygoustransgenic lines of Arabidopsis transgenic plants bearing the construct35S:HaWRKY76.

FIG. 8A shows root lengths of three homozygous transgenic lines ofArabidopsis transgenic plants bearing the construct 35S:HaWRKY76 and ofWT control plants, and the photograph of FIG. 8B shows roots of the7-day-old plants grown on Petri dishes.

FIG. 9 shows the average aerial biomass (weight of detached rosettes andstems) of 35-day-old plants belonging to three homozygous transgeniclines of Arabidopsis transgenic plants bearing the construct35S:HaWRKY76 and of 35-day-old WT control plants that were grown instandard conditions. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 10A shows the weight of rosettes detached from plants belonging tothree homozygous transgenic lines of Arabidopsis transgenic plantsbearing the construct 35S:HaWRKY76 and of the WT control plants grown onpoor soil in standard conditions. FIG. 10B shows the chlorophyll contentper mg of rosette leaves of each of the respective plants. FIG. 10Cshows the total protein concentration of each of the respective plants.Different letters indicate samples that are significantly different (pvalue<0.05).

FIG. 11 shows the root biomass of 35-day-old plants belonging to threehomozygous transgenic lines of Arabidopsis transgenic plants bearing theconstruct 35S:HaWRKY76 and the WT control plants, all grown on sandirrigated with Hoagland 0.5×. Different letters indicate samples thatare significantly different (p value<0.05).

FIG. 12A shows the average seeds yield of 4 plants of each genotypegrown on poor soil conditions (nutritional deficiency), FIG. 12B showsthe yield per individual plant, and FIG. 12C is a photograph of theharvested seeds (35S:HaWRKY76 transgenic seeds and WT control seeds).Different letters indicate samples that are significantly different (pvalue<0.05).

FIG. 13 shows the yields of plants belonging to three homozygoustransgenic lines of Arabidopsis transgenic plants bearing the construct35S:HaWRKY76 and of the WT control plants grown in standard conditions.Different letters indicate samples that are significantly different (pvalue<0.05).

FIG. 14 is a photograph of plants belonging to three homozygous lines ofArabidopsis transgenic plants bearing the construct 35S:HaWRKY76 and ofWT control plants that have been subjected to severe drought stress. Thephotograph is taken 4 days after the plants were re-watered followingthe severe drought stress treatment.

FIG. 15A is a photograph of the plants belonging to three homozygoustransgenic lines of Arabidopsis transgenic plants bearing the construct35S:HaWRKY76 and of WT control plants that were subjected to droughtduring the vegetative stage, and FIG. 15C is a photograph of therespective plants subjected to drought during the reproductive stage.FIG. 15B shows yields of the respective plants subjected to droughtduring the vegetative stage, and FIG. 15D shows yields of the respectiveplants subjected to drought during the reproductive stage. In bothtreatments, a nutritional stress (generated by growing the plants onpoor soil) was applied. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 16A shows the enhanced yield of Arabidopsis transgenic plantsbearing the construct 35S:HaWRKY76 when drought stress was appliedduring the vegetative stage, and FIG. 16B shows that no significantdifferences in yield between the genotypes were observed when thedrought stress was suffered during the reproductive stage. In theseexperiments, the only stress applied was drought and other conditions(soil) were standard. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 17A shows the average water added, and FIG. 17B shows the averageyield per plant for each of the three homozygous transgenic lines ofArabidopsis transgenic plants bearing the construct 35S:HaWRKY76 and theWT control plants. All of the plants were grown on soil and normalwatering was stopped when the plants were 25 days old. Thereafter, theminimum quantity of water was added every two days to maintain the sameweight in all pots. Different letters indicate samples that aresignificantly different (p value<0.05).

FIGS. 18A and 18B show the weight loss of leaves detached from plantsbelonging to three homozygous transgenic lines of Arabidopsis transgenicplants bearing the construct 35S:HaWRKY76 and WT control plants thatwere well-irrigated (FIG. 18A) and subjected to moderate drought stress(FIG. 18B). FIG. 18C is an illustrative photograph of leaves of therespective plants taken after 8 hours of treatment. Different lettersindicate samples that are significantly different (p value<0.05).

FIG. 19A shows the percentage of survivors after 25 day-old plants ofthree homozygous transgenic lines of Arabidopsis transgenic plantsbearing the construct 35S:Ha WRKY76 and WT control plants werecompletely submerged during 6 days and then placed in standard growthconditions for recovery. FIG. 19B is an illustrative photograph of theplants taken one week after recovery. FIG. 19C shows the average seedyields of the recovered plants.

FIG. 19D shows the harvested seeds of each genotype. The number ofsurvivors was lower for the WT genotype plants. Therefore, the totalyield is significantly different (measured as the average) because onlythe survivors contribute. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 20A shows the rosette weight of the respective plants measured 2and 5 days after starting the treatment conditions referenced in FIG.19. FIG. 20B is an illustrative photograph of the rosettes of the plantstaken 5 days after starting the treatment. FIG. 20C shows thechlorophyll content/mg of rosette leaves of the respective plantsmeasured 2 and 5 days after starting the treatment. FIG. 20D shows thecontent of total soluble sugars/mg of rosette leaves that wasenzymatically determined for each of the plants. This content wasevaluated 2 and 5 days after starting the treatment. Different lettersindicate samples that are significantly different (p value<0.05).

FIGS. 21A-C show the content of soluble carbohydrates and starch inrosettes of 25 day-old plants of three homozygous transgenic lines ofArabidopsis transgenic plants bearing the construct 35S:HaWRKY76 and ofWT control plants. The 25-day old plants were submerged during 0, 2, 5days. FIG. 21A shows the content of soluble glucose per mg fresh rosetteweight of the respective plants, FIG. 21B shown the sucrose content permg fresh rosette weight of the respective plants, and FIG. 21C shows thestarch content per mg fresh rosette weight of the respective plants.Different letters indicate samples that are significantly different (pvalue<0.05).

FIG. 22A shows the chlorophyll content per mg fresh rosette weight of 25day-old plants of three homozygous transgenic lines of Arabidopsistransgenic plants bearing the construct 35S:HaWRKY76 and of WT controlplants. FIG. 22B shows the protein content per mg fresh rosette weightof the respective plants. The respective plants were completelysubmerged during 5 days. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 23A shows the root/aerial biomass ratio of 35 day-old plants ofthree homozygous transgenic lines of Arabidopsis transgenic plantsbearing the construct 35S:HaWRKY76 and of WT control plants. FIG. 23Bshows the root protein content of the respective plants. The respectiveplants were grown on sand, and on day 35 the complete root system wascollected and weighed. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 24 includes photographic images of 25 day-old plants of threehomozygous transgenic lines of Arabidopsis transgenic plants bearing theconstruct 35S:Ha WRKY76 and of WT control plants. The respective plantswere subjected to waterlogging during 5 days, and transverse sections ofstems were performed and stained.

FIG. 25 shows the average glucose content obtained from 4 plants of eachgenotype (three homozygous transgenic lines of Arabidopsis transgenicplants bearing the construct 35S:HaWRKY76 and WT control plants)evaluated both after 5 days of complete submergence and one day afterrecovery. Different letters indicate samples that are significantlydifferent (p value<0.05).

FIG. 26 shows the average yield (at the end of the life cycle) of25-day-old plants of each genotype (three homozygous lines ofArabidopsis transgenic plants bearing the construct 35S:HaWRKY76 and WTcontrol plants) that were grown in standard growing conditions, thencompletely submerged during 5 days, and then recovered. Differentletters indicate samples that are significantly different (pvalue<0.05).

FIG. 27A shows rosette weight measured 6 and 7 days after waterloggingwas applied during a week in 25-day-old plants of each genotype grown onpoor soil conditions. FIG. 27B shows the membrane stability of therespective plants, evaluated 5 and 7 days after the start ofwaterlogging. FIG. 27C shows the stem length of the respective plantsafter waterlogging. Different letters indicate samples that aresignificantly different (p value<0.05).

FIGS. 28A and 28B show the average yield and the yield of each plant atthe end of the life cycle, respectively, after one week of thewaterlogging treatment that was applied to 25-day old plants asdescribed in FIG. 27. Different letters indicate samples that aresignificantly different (p value<0.05).

FIG. 29 shows the average yield per genotype (three homozygoustransgenic lines of Arabidopsis transgenic plants bearing the construct35S:Ha WRKY76 and WT control plants) that were subjected to awaterlogging treatment during one week after being grown in standardsoil. Different letters indicate samples that are significantlydifferent (p value<0.05).

FIG. 30 shows the number of rosette leaves exhibited by plants belongingto three homozygous transgenic lines of Arabidopsis transgenic plantsbearing the construct 35S:Ha WRKY76 and WT control plants that weregrown in standard conditions. Different letters indicate samples thatare significantly different (p value<0.05).

FIG. 31 shows the stem lengths measured during the life cycle of plantsbelonging to three homozygous transgenic lines of Arabidopsis transgenicplants bearing the construct 35S:HaWRKY76 and WT control plants.Different letters indicate samples that are significantly different (pvalue<0.05).

FIG. 32 is an image of an EMSA assay performed with purified recombinantHaWRKY76-GST. 12N is a double stranded oligonucleotide exhibiting arandom sequence whereas W-Box is a double stranded oligonucelotidehaving the canonical W-box.

FIG. 33 is a schematic representation of three kinds of assays withdifferent water levels: a) hardly on the substrate, b) 1 cm above therosette leaves, and c) 2-4 cm below the basal portion of the maininflorescence.

FIGS. 34A-C show HaWRKY76 expression in sunflower. FIG. 34A shows5-day-old seedlings well irrigated (control panel) or exposed to severewater stress (drought panel). FIG. 34B shows 15-day-old plantlets (time0) exposed to severe drought stress and FIG. 34C shows submergenceduring 11 days and recovery (R). Transcript levels of HaWRKY76 werequantified by RT-qPCR, normalized with sunflower actin (ACTIN2 andACTIN8), and thereafter with respect to the value (FIG. 34A) measured inthe cotyledon sample under control conditions, (FIG. 34B) in the sampleexposed to water stress during 2 days, or (FIG. 34C) in the beginning ofthe treatment, the three arbitrarily assigned a value of one. Twoindependent experiments were done and error bars correspond to standarddeviations from three biological replicas in each experiment. An ANOVAtest was performed, followed by a Fisher LSD post-hoc test. Differentnumbers of asterisks indicate samples with significant differences(P<0.05).

FIGS. 35A-D show HaWRKY76 transgenic plants exhibit equal or higheryield than WT after water deficit or water excess treatments. FIGS. 35Aand 35B show seed production of transgenic (W76-A, W76-B, W76-C) and WTplants subjected to mild water deficit in the vegetative (FIG. 35A) orthe reproductive (FIG. 35B) stage. FIGS. 35C and 35D show seedproduction of transgenic (W76-A, W76-B, W76-C) and WT plants in thereproductive stage subjected to submergence (FIG. 35C) or waterlogging(FIG. 35D). Two or three plants per pot were assayed. Three experimentswere done and error bars correspond to standard deviations from fourbiological replicas in each experiment. ANOVA test was performed,followed by a Fisher LSD post-hoc test. Asterisks indicate samples whichare significantly different from WT (P<0.05)

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION Definitions

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation, and amino acid sequences are written left to rightin amino to carboxy orientation, respectively. Numeric ranges areinclusive of the numbers defining the range and include each integerwithin the defined range. Amino acids may be referred to herein byeither their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes.

The term “amplified” refers to the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequence as atemplate. Amplification systems include, but are not limited to,polymerase chain reaction (PCR), ligase chain reaction (LCR) system,nucleic acid sequence based amplification (NASBA), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., D. H. Persing et al.,“Diagnostic Molecular Microbiology: Principles and Applications,”American Society for Microbiology, Washington D.C. (1993).

The term “introduced” or “introducing” as used herein in the context ofinserting into a cell refers to the incorporation of a nucleic acid intoa target cell, such as a plant cell, such that the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). In embodiments,introducing a nucleotide sequence into a plant cell results intransformation of the plant cell to cause stable or transient expressionof the sequence.

The term “isolated” as used herein refers to material, such as a nucleicacid or a protein, which is substantially or essentially free ofcomponents that normally accompany or interact with the material withinits naturally occurring environment. The isolated material may include amaterial not found with the material in its natural environment, or ifthe material is in its natural environment, the material has beensynthetically (i.e., non-naturally) altered by deliberate humanintervention to form a composition and/or be found in a location in thecell (e.g., genome or subcellular organelle) not native to the materialfound in that environment. The alteration forming the synthetic materialcan be performed on the material within or removed from its naturalstate. For example, a naturally occurring nucleic acid becomes anisolated nucleic acid if it is altered, by means of human interventionon the cell from which it originates. Likewise, a naturally occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced bynon-naturally occurring means to a locus of the genome not native tothat nucleic acid, as discussed further below.

As used herein, the term “nucleic acid” (or “polynucleotide”) refers toa deoxyribonucleotide or a ribonucleotide polymer, or analog thereof,that has the essential nature of natural nucleotides in that ithybridizes, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallows translation into the same amino acid(s) as the naturallyoccurring nucleotide(s). A polynucleotide can be full-length or asub-sequence of a native or heterologous structural or regulatory gene.Unless otherwise indicated herein, the term refers to a specifiedsequence, as well as the complementary sequence thereof. Thus, DNAs orRNAs with backbones modified for stability or for other reasons, as wellas DNAs and RNAs comprising unusual or modified bases, arepolynucleotides as the term is defined herein. The term polynucleotidealso encompasses chemically, enzymatically, or metabolically modifiedforms of polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of simple and complex cells. The term “nucleic acid” (or“polynucleotide”) may be used in place of, inter alia, gene, cDNA, mRNA,or cRNA.

As used herein, the term “operably linked” refers to a functionallinkage between sequences, such as a promoter and a second sequence,wherein the promoter sequence initiates and mediates transcription ofthe DNA sequence corresponding to the second sequence. Generally,operably linked means that the nucleic acid sequences being linked arecontiguous and, where necessary to join two protein coding regions,contiguous in the same reading frame.

The term “plant” is used broadly herein to describe a plant at any stageof development, to a part of a plant (e.g., plant cell, plant cellculture, plant organ, plant seed, etc.), and to progeny thereof. A“plant cell” is the structural and physiological unit of the plant,comprising a protoplast and a cell wall. A plant cell can be in the formof an isolated single cell or a cultured cell, or can be part of ahigher organized unit, such as plant tissue, a plant organ, or a plant.Thus, a plant cell can be a protoplast, a gamete-producing cell, or acell or collection of cells that can regenerate into a whole plant. Asused herein, a “seed” comprises multiple plant cells and is capable ofregenerating into a whole plant, and may therefore be considered a plantcell. A plant tissue or organ can be a seed, protoplast, callus, or anyother group of plant cells that is organized into a structural orfunctional unit. Parts of a plant that are particularly useful inembodiments include harvestable parts and parts used for propagation ofprogeny plants. A harvestable part of a plant may include the flowers,pollen, seedlings, tubers, leaves, stems, fruit, seeds, roots, and thelike. Parts of the plant used for propagation include, e.g., seeds,fruits, cuttings, seedlings, tubers, rootstocks, and the like. The classof plants that may be used is generally as broad as the class of higherplants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants.

The terms “polypeptide” and “protein” as used herein refer to a polymerof amino acid residues. The terms encompass amino acid polymers, inwhich one or more amino acid residue is an artificial chemical analogueof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The essential nature of suchanalogues of naturally occurring amino acids is that, when incorporatedinto a protein, the protein is specifically reactive to antibodieselicited to the same protein but consisting entirely of naturallyoccurring amino acids. The polypeptide group includes, but is notlimited to, DNA binding proteins, protein kinases, protein phosphatases,GTP-binding proteins, and receptors.

As used herein, the term “promoter” refers to a region of DNA that isupstream from the start of transcription and that is involved inrecognition and binding of RNA polymerase and other proteins to initiatetranscription. The promoter may be any polynucleotide sequence thatshows transcriptional activity in the host (target) plant cells, plantparts, or plants.

As used herein, the term “recombinant” refers to a cell or vector thathas been modified by the introduction of a heterologous nucleic acid ora cell that is derived from a cell so modified. For example, recombinantcells express genes that are not found in identical form within thenative (non-recombinant) form of the cell or express native genes thatare otherwise abnormally expressed, under-expressed, or not expressed atall as a result of deliberate human intervention. The term does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, or natural transformation,transduction, or transposition).

As used herein, the term “recombinant expression cassette” (or“expression cassette”) refers to a nucleic acid construct that isrecombinantly or synthetically generated with a series of specifiednucleic acid elements, that permits transcription of a particularnucleic acid in a target cell. The recombinant expression cassette canbe incorporated into a plasmid, chromosome, mitochondrial DNA, plastidDNA, virus, or nucleic acid fragment. Typically, the recombinantexpression cassette portion of an expression vector includes a nucleicacid to be transcribed and a promoter.

As used herein, the term “regulatory element” means a nucleotidesequence that, when operatively linked to a coding region of a gene,effects transcription of the coding region such that a ribonucleic acid(RNA) molecule is transcribed from the coding region. Regulatoryelements include promoters, enhancers, silencers, 3′-untranslated or5′-untranslated sequences of transcribed sequences, e.g., a poly-Asignal sequence or other protein or RNA stabilizing element, or othergene expression control elements known to regulate gene expression orthe amount of expression of a gene product.

The terms “residue”, “amino acid residue”, and “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide. The amino acid may be anaturally occurring amino acid and, unless otherwise limited, mayencompass non-natural analogs of natural amino acids that can functionin a similar manner as naturally occurring amino acids.

As used herein, “sequence identity” in the context of two polynucleotideor polypeptide sequences refers to the residues in the two sequencesthat are the same when aligned for maximum correspondence over acomparison window of a contiguous and specified segment of apolynucleotide sequence. When percentage of sequence identity is used inreference to proteins, it is recognized that residue positions that arenot identical often differ by conservative amino acid substitutions.Where sequences differ in conservative substitutions, the percentsequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences differing by suchconservative mutations are said to have “sequence similarity.” Methodsfor making this adjustment are well known to persons skilled in the art.The “percentage” of sequence identity means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may include additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity.

As used herein, the term “transgenic plant” refers to a plant thatincludes within its genome a heterologous polynucleotide. Generally, theheterologous polynucleotide is stably integrated within the genome ofthe transgenic plant, such that the polynucleotide is passed on tosuccessive generations. The term “transgenic” is used herein to describeany cell, cell line, callus, tissue, plant part or plant (also referredto herein as a “target cell” or “host cell”) the genotype of which hasbeen altered by the presence of a heterologous nucleic acid, andincludes transgenic plants that have been initially altered, as well asthose created by sexual crosses or asexual propagation from the initialtransgenic plants. The term “transgenic” as used herein does notencompass the alteration of the genome by naturally occurring events,such as random cross-fertilization or spontaneous mutation.

As used herein, the term “vector” refers to a nucleic acid used in thetransfection of a target cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

As used herein, the term “wild-type” (or “WT”) refers to a cell or plantthat has not been genetically modified to knock out or over-expresspolypeptides according to embodiments of the present disclosure.Wild-type cells or plants may be used as controls to compare levels ofexpression and the extent and nature of trait modification ingenetically modified (i.e., transgenic) cells or plants in whichpolypeptide expression is altered or ectopically expressed by, forexample, knocking out or over-expressing a gene.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention will now be further described. In the followingpassages, different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, bioinformatics which are within the skill of the art. Suchtechniques are explained fully in the literature.

External stress factors, from both biological and abiotic origins,affect the levels of specific proteins by transcriptional and/orpost-transcriptional regulation. The ability of a given species tosurvive various stress conditions is intimately related to a series ofmolecular responses involving activation and repression of certaingenes. The stress tolerance of a species appears to be controlledprimarily at the transcriptional level, depending on the transcriptionfactor activity.

As in other species, the primary players regulating gene expression insunflowers are transcription factors and genomic regulatory regions (aswell as small RNAs, including miRNAs). Transcription factors areproteins that are able to recognize and bind specific DNA sequencespresent in the regulatory regions of their target genes, and modulatetheir transcription. It is known that transcription factors have amodular structure and exhibit at least two types of domains: a DNAbinding domain; and a protein-protein interaction domain, which mediates(directly or indirectly) the activation or repression of transcription.See Brivanlou and Darnell, “Signal transduction and the control of geneexpression,” Science 295:813-818 (2002). Additionally, transgenic plantscomprising isolated polynucleotides encoding transcription factors mayalso modify expression of endogenous genes, polynucleotides, andproteins. See Peng et al., Genes and Development 11:3194-3205 (1997),and Peng et al., Nature 400:256-261 (1999).

Within a single plant species, gene duplication may cause two copies ofa particular gene, giving rise to two or more genes with similarsequences and often similar functions, known as paralogs. A paralog istherefore a similar gene formed by duplication within the same species.Paralogs typically cluster together or in the same clade (i.e., a groupof similar genes) when a gene family phylogeny is analyzed usingprograms known to persons skilled in the art. For example, a clade ofvery similar transcription factors of the same species typically willshare a common function (e.g., flowering time, drought tolerance, etc.).Analysis of groups of similar genes having a similar function that fallwithin one clade can yield sub-sequences that are particular to thatclade. These sub-sequences, known as “consensus sequences,” can be usednot only to define the sequences within each clade, but also to definethe functions of these genes; genes within a clade may containparalogous sequences, or orthologous sequences that share the samefunction. See, e.g., Mount, “Bioinformatics: Sequence and GenomeAnalysis,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., p. 543 (2001).

Transcription factor gene sequences are conserved across diverseeukaryotic species lines. Plants are no exception to this observation;diverse plant species possess transcription factors that have similarsequences and functions. Speciation for the production of new speciesfrom a parental species gives rise to two or more genes with similarsequence and similar function. These genes, or “orthologs,” often havean identical function within their host plants and are ofteninterchangeable between species without losing function. Because plantshave common ancestors, many genes in any plant species will have acorresponding orthologous gene in another plant species. Once aphylogenic tree for a gene family of one species has been construedusing a program, such as CLUSTAL, potential orthologous sequences can beplaced into the phylogenetic tree and their relationship to genes fromthe species of interest can be determined. Orthologous sequences canalso be identified by a reciprocal BLAST strategy. Once an orthologoussequence has been identified, the function of the ortholog can bededuced from the identified function of the reference sequence. By usinga phylogenetic analysis, persons skilled in the art would be able topredict similar functions conferred by closely-related polypeptides. Anorthologous sequence of a plant, including plants specifically mentionedherein, can have at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the Ha nucleic acid or protein described herein.The invention also includes embodiments that relate to orthologoussequences.

Although approximately 2000 plant transcription factors have beenidentified in plants in silico and classified in families andsub-families according to similarities in the respective bindingdomains, gene structures, functions and other structural features, onlya small percentage of those transcription factors has been functionallycharacterized (even in model plants). The WRKY transcription factors ofArabidopsis are an example of one such classified family, which has alsobeen functionally characterized as related to abiotic and biotic stressresponses. See, e.g., Giacomelli et al. (2010) and Giacomelli et al.(2012). Specifically, the distinct transcription factors of the WRKYfamily in Arabidopsis and numerous closely-related sequences from dicotsand monocots have been shown to confer increased water deprivationtolerance.

The WRKY transcription factors are primarily characterized by thepresence of a 60 amino acid conserved region containing the four WRKYamino acids and a zinc-finger-like motif, which together form the WRKYdomain. These proteins are unique to plants and have been classified inArabidopsis into three main groups (I, II, and III) on the basis of boththe number of WRKY domains and the pattern of the zinc-finger-likemotif, with the second group being further classified in five subgroups(IIa, IIb, IIc, IId, and IIe). See Eulgem and Somssich, “Networks ofWRKY transcription factors in defense signaling,” Curr. Opin. PlantBiol. 10:366-371 (2007); and Rushton et al., “WRKY transcriptionfactors,” Trends in Plant Sci., 15:247-258 (2010).

In a study to identify and characterize WRKY transcription in thesunflower (belonging to the Asteraceae family) using in silicoapproaches and gene expression surveys, a bioinformatics analysis of ESTdatabases estimated the existence of 97 sunflower WRKY members. SeeGiacomelli et al., “Expression analyses indicate the involvement ofsunflower WRKY transcription factors in stress responses, andphylogenetic reconstruction reveal the existence of a novel clade in theAsteraceae,” Plant Science 178:398-410 (2010). Phylogenetic treesconstructed with WRKY domains resolved the same seven groups assigned toArabidopsis, as well as a novel clade diverging with the IId subgroup asdescribed above, with traits apparently specific to Asteraceae. The 2010study referenced above indicated that the WRKY family has undergone aparticular diversification, which could be the source of specific newfunctions. A partial sequence of HaWRKY76, a member of this Asteraceaespecific clade, was also reported in the study.

A schematic representation of the deduced proteins with a WKKY motifcorresponding to EST-clusters from Helianthus spp. and Lactuca spp. isprovided in Giacomelli et al. (2010), which shows the putative conservedprimary structural features of the Helianthus/Lactuca WKKY(WKKYGEK, SEQID NO:7) motif encoding proteins. All of the expressed sequence tags(ESTs) identified as proteins having WKKY motif-encoding sequences (52)were obtained from GenBank, and the EST-clusters resulting from theassembly (18) were translated. In Giacomelli's schematic representation,conserved primary structural features identified by using MEME (Baileyand Elkan (1994)) are identified with grey boxes and labels. Box A showsthe N-terminal consensus sequence, box H shows the HARF motif, box Cshows the calmodulin-binding protein, box Z shows the zinc cluster, andbox P shows the proline-rich motif. An alignment was performed usingMAFFT (Lopez R. (1997)) and two regions from it are shown in detailbelow the scheme described above. The first region shown is theserine-rich region, which has divergences in five identified clusters.The second region shows is the WKKY domain.

The WKKY clade proteins present structural differences relative tomembers of the WRKY family. Specifically, the WKKY proteins containmotifs unique to the IId WRKY members, the C, the HARF and a zinccluster upstream of the WKKY domain (not the zinc-finger-like motifproper of the WRKY domain). Moreover, they exhibit an additionalremarkable feature: the presence of two substitutions in the main regionof the WRKYGQK (SEQ ID NO:8) domain that result in a WKKYGEK motif (SEQID NO:7). Other signatures that have not been reported in other WRKYproteins, such as a conserved N-terminal region, a putativeserine-threonine kinase domain, and proline-rich region (P), were alsoclearly identified.

Since WKKY proteins share conserved structures with IId WRKY groupmembers, it is possible that they have some properties in common. Forexample, the expression of members of the IId WRKY group is induced bypathogen infection and SA. Additionally, EMSA assays performed withAtWRKY11 showed that the substitution of RK by KR in the WRKY domainavoided the interaction with the W-box. See Ciolkowski et al., “Studieson DNA-binding selectivity of WRKY transcription factors lend structuralclues into WERKY-domain function,” Plant Molecular Biology 68:81-92(2008). In contrast, EMSA assays performed with HaWRKY76 indicated thatthis protein selected a different DNA sequence. Because WKKY proteinsshare conserved structures with IId WRKY members, it is possible thatthe proteins have in common the properties associated with thesestructures. Similar to the expression of proteins belonging to the IIdWRKY group being induced by pathogen infection and SA concomitantly witha calcium discharge, the zinc cluster could be involved in determiningthe DNA affinity as it was demonstrated for AtWRKY11. However, thisArabidopsis transcription factor presented a reduced affinity for aW-box when a single D to E amino acid was substituted within theN-terminal region to the WRKY domain. Because sunflower proteins with aWKKY motif do not have a D within the zinc cluster, different aminoacids could be determining the affinity of these proteins for theirtarget sequences.

The expression of sunflower WRKY genes is regulated by hormones, abioticand biotic factors, and wounding. The published study of Giacomelli etal. (2010) includes a comparative diagram of selected expressionprofiles of sunflower WRKY genes after three-hour treatments withhormones or factors associated with stress, i.e., the hormones CK (100μM 6-benzylaminopurine), GA (100 μM gibberellin A3), AUX (100 μMindole-3-acetic acid), ACC (30 μM aminocyclopropane-carboxylic acid), JA(200 μM MeJA), and SA (1 mM), the wounding damage (WO), MAN 350 mMD-mannitol), SALT (150 mM NaCl) and the Pseudomonas syringae DC3000spraying (PS). The transcript levels were measured by quantitativeRT-PCR and standard errors were calculated from three biologicalreplicates in which actin transcripts (HaACTIN) were used as internalcontrols.

The presence of the exclusive stretches found only in members of theWKKY clade suggests that they could be the basis of a neo-functionality.In this sense, the serine-rich sequence found by the ELM program couldbe a target of a serine-threonine kinase. PPLP motifs are proline-richsequences present in ligands interacting with WW domains. Notably,proline residues in the PPLP motif identified in sunflower WKKYs arequite equally distributed as in FY PPLP [PxP(x)₇PPLP], but the spacersequences are completely different.

Besides the 2010 publication and the subsequent Ph.D. Thesis of JorgeGiacomelli (2011), no additional information regarding any HaWRKY76sequences (including the HaWRKY76 sequence of Giacomelli (2010)) havebeen reported, and no functional analyses have been conducted.

Using techniques described herein, the inventors have now cloned thecomplete HaWRKY76 under the control of a 35S CaMV promoter. The HaWRKY76nucleotide sequence and polypeptide (protein) sequence below correspondto SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The HaWRKY76transcription factor has a WKK motif because there is a change of R to Kin the WRKY domain. Thus, HaWRKY76 is also referred to as a WKKYtranscription factor herein.

HaWRKY76 nucleotide sequence of SEQ ID NO: 1  (cDNA)atggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttatcacattcggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgtttttcaaacaaaccggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagccaaagtgaaCorresponding HaWRKY76 polypeptide (protein)  sequence of SEQ ID NO: 2MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHARFRRGPSSTTGDTNGPSTSSHSEGKSRDTTSFVQNECFSNKPVTEITTTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSSLGIVAPAPTFSSRKPPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVPITGSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARKRVELSADDSKMLIVTYDGEHRHRDRHAPVPMSLTGVYGEPK

A sequence having similarity with the HaWRKY76 sequences disclosedherein has been reported in the Helia database. The gene and proteinsequences of HaT131007971 correspond to SEQ ID NO: 3 and SEQ ID NO: 4,respectively. A comparison of the HaT131007971 polypeptide (protein)sequence (SEQ ID NO: 4) with the full-length polypeptide (protein)sequence of HaWRKY76 (SEQ ID NO: 2) indicates several differences, asshown in FIG. 1. A comparison of the HaT131007971 polynucleotidesequence (SEQ ID NO: 3) with the full-length polynucleotide sequence ofHaWRKY76 (SEQ ID NO: 1) indicates several differences, as shown in FIG.2.

Another sequence having similarity with the HaWRKY76 sequences disclosedherein is HuCL13748C001, which has also been reported in the Heliadatabase. The gene and protein sequences of the HuCL13748C001 correspondto SEQ ID NO: 5 and SEQ ID NO: 6, respectively. A comparison of thepolypeptide (protein) sequence of HuCL13748C001 (SEQ ID NO: 6) with thefull-length polypeptide (protein) sequence of HaWRKY76 (SEQ ID NO: 2)indicates several differences, as shown in FIG. 3. A comparison of thepolynucleotide sequence of HuCL13748C001 (SEQ ID NO: 5) and thefull-length polynucleotide sequence of HaWRKY76 (SEQ ID NO: 1) indicatesseveral differences, as shown in FIG. 4. These differences have not yetbeen evaluated for functional roles, but some of them are notconservative and/or located in putative functional domains.

It is generally understood that the up-regulation of a certain gene byany abiotic stress factor does not mean that the gene will confertolerance to such stress if it is used as a transgene. Moreover, theability of a gene to confer tolerance to a given stress factor does notimply that it will confer tolerance to other stress factors. Manyexamples of genes conferring tolerance to drought but not to, e.g., hightemperatures, are described in scientific literature and understood bypersons skilled in the art. In the same way, tolerance to lowtemperatures above 0° C. (chilling) is generally not concomitant withtolerance to low temperatures below 0° C. (freezing) since differentmolecular mechanisms are playing a role in these responses.

In the same way, it is also generally understood that tolerance todrought does not imply tolerance to submergence or waterlogging becausedifferent signal transduction pathways are triggered in these responses.Likewise, tolerance to drought or to any other abiotic stress factordoes not imply increased yield under such stresses. While tolerance isusually evaluated and reported as a percentage of survivors after asevere stress treatment, the yield under such conditions is generallynot informed under moderate stress conditions. See e.g., Skirycz et al.,“Survival and growth of Arabidopsis plants given limited water are notequal,” Nature Biotechnology 29:212-214 (2011) (reporting that 25 genesknown to confer drought tolerance exhibited decreased yield in standardconditions or under a moderate drought stress). Such moderate stress isthe most common and probable growing condition encountered by plants inthe field.

Thus, a combination of increased yield (or at least no decrease inyield) and stress tolerance represents a very valuable characteristic oftechnologies to improve crops. As described in connection with thevarious embodiments and specific Examples provided herein, HaWRKY76unexpectedly offers this highly advantageous combination ofcharacteristics.

As indicated above, the complete HaWRKY76 was cloned under the controlof the 35S CaMV promoter. This construct was further used to transformArabidopsis plants to produce transgenic plants exhibiting differentexpression levels of HaWRKY76. Plants produced according to the methodsdescribed herein were analyzed both in standard growth conditions and ingrowth conditions in which they were subjected to abiotic stressfactors.

The analysis revealed, among other things, that when grown understandard conditions, 35S:HaWRKY76 transgenic plants have a similarnumber of rosette leaves, life cycle duration and stem length as thecorresponding WT control plants. However, transgenic plants bearing theconstruct 35S:HaWRKY76 were found to have longer roots and largerrosettes than corresponding WT control plants. Moreover, total proteinand chlorophyll contents of the transgenic plants were found to beproportional to the rosette weight (i.e., 35S:HaWRKY76 plants producemore biomass and protein than control plants). Furthermore, whensubjected to water stress, transgenic plants bearing the construct35S:HaWRKY76 were found to be more tolerant to drought thancorresponding WT control plants, and similar properties were observedwhen the transgenic plants were stressed by submergence or waterlogging.Notably, transgenic plants bearing the construct 35S:HaWRKY76 not onlydemonstrated improved tolerance to the various moderate and severestress conditions, but also exhibited higher yields than thecorresponding WT control plants (yield evaluated as a measure of seedproduction).

The HaWRKY76 transcription factor polypeptides confer on transgenicplants produced according to methods described herein improved stresstolerance, as well as increased yield in standard growing conditions.Specifically, as evidenced by the various Examples and applicableFigures, the inventors unexpectedly discovered that the HaWRKY76sequences confer tolerance to drought, submergence and waterlogging,while also increasing yield in standard conditions.

It will be understood by persons skilled in the art that embodiments ofthe invention also relate to, among other things, the isolation andfunctional characterization of the HaWRKY76 nucleotide and amino acidsequences, transgenic plants transformed with constructs comprising theHaWRKY76 polynucleotide sequences, and methods of producing transgenicplants expressing the HaWRKY76 polypeptide sequences disclosed herein,wherein the transgenic plants have improved stress tolerance andincreased yield in comparison to corresponding control plants, forexample WT plants.

WRKY76 Transcription Factor Polynucleotides

In embodiments of the aspects of the invention, the polynucleotidesdescribed herein include nucleotide sequences that encode WRKY76transcription factors and transcription factor homolog polypeptides andsequences complementary thereto, as well as unique fragments of a codingsequence, or a sequence complementary thereto. The polynucleotides maybe, e.g., DNA or RNA, such as mRNA, cRNA, synthetic RNA, genomic DNA,cDNA synthetic DNA, oligonucleotides, etc. The polynucleotides areeither double-stranded or single-stranded and include either or bothsense (i.e., coding) sequences and antisense (i.e., non-coding,complementary) sequences. The polynucleotides may include the codingsequence of a transcription factor or transcription factor homologpolypeptide, in isolation, in combination with additional codingsequences, in combination with non-coding sequences (e.g., introns,regulatory elements such as promoters, enhancers, terminators, and thelike), and/or in a vector or host environment in which thepolynucleotide encoding a transcription factor or transcription factorhomolog polypeptide is an endogenous or exogenous gene. WRKY76transcription factors include the signature motif WKKYGEK (SEQ ID NO:7).WRKY76 transcription factors also include a conserved serine-threoninekinase domain and a proline-rich region.

Representative polynucleotides of WRKY76 transcription factors includethe full-length polynucleotide sequence of SEQ ID NO: 1, and functionalvariants or parts thereof which retain biological function of thefull-length polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9, forexample SEQ ID NO: 11. Preferably, variants are substantially identicalpolynucleotides. Substantially identical polynucleotides comprisenucleotide sequences that vary from the full-length amino acid sequenceof SEQ ID NO: 1 by one or more modifications, including deletions,substitutions, or additions, the net effect of which is retainedbiological function of the WRKY76 polynucleotide. For example,substantially identical polynucleotides comprise nucleotide sequencesthat vary from the full-length amino acid sequence of SEQ ID NO: 1 by 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, or additions. In someembodiments, variants, such as substantially identical WRKY76polynucleotides may have at least 80% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9 asdescribed herein, or by visual inspection. Preferably, the WRKY76polynucleotides have at least 85% sequence identity, or at least 90%sequence identity, or at least 91% sequence identity, or at least 92%sequence identity, or at least 93% sequence identity, or at least 94%sequence identity, or at least 95% sequence identity, or at least 96%sequence identity, or at least 97% sequence identity, or at least 98%sequence identity, or at least 99% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1. For example, sequenceidentity is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8′7%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. A variantwithin the scope of the various aspects of the invention is shown in SEQID NO: 11 and the corresponding polypeptide SEQ ID NO: 12.

In some embodiments of the various aspects of the invention, theisolated polynucleotides of the invention contain at least one of thefollowing: (a) adenine as the nucleotide corresponding to position 817of the full-length nucleotide sequence of SEQ ID NO: 1 or a variantthereof, for example SEQ ID NO: 11; (b) cytosine as the nucleotidecorresponding to position 808 of the full-length nucleotide sequence ofSEQ ID NO: 1 or a variant thereof, for example SEQ ID NO: 11; (c)cytosine as the nucleotide corresponding to position 33 of thefull-length nucleotide sequence of SEQ ID NO: 1 or a variant thereof,for example SEQ ID NO: 11; (d) cytosine as the nucleotide correspondingto position 259 of the full-length nucleotide sequence of SEQ ID NO: 1or a variant thereof, for example SEQ ID NO: 11; and (e) guanine as thenucleotide corresponding to position 315 of the full-length nucleotidesequence of SEQ ID NO: 1 or a variant thereof, for example SEQ ID NO:11. In particular, in one aspect, the invention relates to an isolatedpolynucleotide having at least 80% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1 or a variant thereof,for example SEQ ID NO: 11, wherein the polynucleotide contains at leastone of:

-   -   (a) adenine as the nucleotide corresponding to position 817 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO: 11;    -   (b) cytosine as the nucleotide corresponding to position 808 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO: 11;    -   (c) cytosine as the nucleotide corresponding to position 33 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO: 11;    -   (d) cytosine as the nucleotide corresponding to position 259 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO: 11; and    -   (e) guanine as the nucleotide corresponding to position 315 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO: 11. Preferably, the isolated        polynucleotide is cDNA.

Substantially identical polynucleotide sequences may be polymorphicsequences, i.e., alternative sequences or alleles in a population inwhich the allelic difference may be as small as one base pair.Substantially identical polynucleotides may also comprise mutagenizedsequences, including sequences comprising silent mutations. A mutationmay comprise one or more residue changes, a deletion of one or moreresidues, or an insertion of one or more additional residues.

Representative polynucleotides according to some embodiments of thevarious aspects of the invention include the polynucleotide comprisingor consisting of SEQ ID NO: 1 or SEQ ID NO: 9 and substantiallyidentical nucleotides, and polynucleotides that encode the HaWRKY76transcription factor polypeptide comprising or consisting of SEQ ID NO:2. Thus, in one embodiment, the isolated polynucleotide comprises orconsists of SEQ ID NO:1. In one embodiment, the isolated polynucleotidecomprises or consists of SEQ ID NO:1.

Substantially identical polynucleotides according to embodiments includepolynucleotides that hybridize specifically to or hybridizesubstantially to the full-length nucleotide sequence of SEQ ID NO: 1, 11or 9 under stringent conditions. In the context of nucleic acidhybridization, two nucleotide sequences being compared may be designatedas a probe and a target. A probe is a reference nucleic acid molecule,and a target is a test nucleic acid molecule, often found within aheterogeneous population of nucleic acid molecules. In this respect, atarget sequence is synonymous with a test sequence.

In some embodiments of the aspects of the invention, the polynucleotidesinclude primers and primer pairs that allow specific amplification ofthe disclosed polynucleotides or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Primers may also beused as probes and can be labeled with a detectable marker, such as, forexample, a radioisotope, fluorescent compound, bioluminescent compound,a chemiluminescent compound, metal chelator or enzyme. A particularnucleotide sequence employed for hybridization studies or assays mayinclude probe sequences that are complementary to at least about 14-40nucleotide sequence of a nucleic acid molecule described herein. Probesmay comprise 14-20 nucleotides, or even longer where desired, such as30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the fulllength of SEQ ID NO: 1, 11 or SEQ ID NO: 9. Such fragments may bereadily prepared, for example by chemical synthesis of the fragment, byapplication of nucleic acid amplification technology, or by introducingselected sequences into recombinant vectors for recombinant production.

Specific hybridization refers to the binding, duplexing, or hybridizingof a molecule only to a particular nucleotide sequence under stringentconditions when that sequence is present in a complex nucleic acidmixture (e.g., total cellular DNA or RNA). Specific hybridization mayaccommodate mismatches between the probe and the target sequencedepending on the stringency of the hybridization conditions.

Stringent hybridization conditions and stringent hybridization washconditions in the context of nucleic acid hybridization experiments,such as Southern and Northern blot analysis, are both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2,Elsevier, New York, N.Y. (1993). Generally, stringent hybridization andwash conditions are selected to be about 5° C. below the thermal meltingpoint for the specific sequence at a defined ionic strength and pH.Typically, under stringent conditions a probe will hybridizespecifically to its target sequence, but not to other sequences.

The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Stringent conditions are selected to be equal to the Tm for a particularprobe. An example of stringent hybridization conditions for Southern orNorthern Blot analysis of complementary nucleic acids having more thanabout 100 complementary residues is overnight hybridization in 50%formamide with 1 mg of heparin at 42° C. An example of highly stringentconditions is 15 minutes in 0.1×SSC at 65° C., whereas an example ofstringent wash conditions is 15 minutes in 0.2×SSC buffer at 65° C. SeeSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Typically, ahigh stringency wash is preceded by a low stringency wash to removebackground probe signal. An example of medium stringency conditions fora duplex of more than about 100 nucleotides is 15 minutes in 1×SSC at45° C. An example of low stringency for a duplex of more than about 100nucleotides is 15 minutes in 4 to 6×SSC at 40° C. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1M Na⁺ ion, typicallyabout 0.01 to 1M Na⁺ ion concentration (or other salts) at pH 7.0-8.3,and the temperature is typically at least about 30° C. Stringentconditions may also be achieved with the addition of destabilizingagents, such as formamide. Additional variations of these conditionswill be readily apparent to those skilled in the art.

Stringency conditions may be selected such that an oligonucleotide thatis perfectly complementary to the coding oligonucleotide hybridizes thecoding oligonucleotide with at least about 5 to 10 times higher signalto noise ratio than the ratio for hybridization of the perfectlycomplementary oligonucleotide to a nucleic acid encoding a transcriptionfactor know in the art. It may be desirable to select conditions for aparticular assay such that a higher signal to noise ratio (e.g., about15× or more) is obtained. Accordingly, a subject nucleic acid willhybridize to a unique coding oligonucleotide with at least 2 times (2×)or greater signal to noise ratio as compared to hybridization of thecoding oligonucleotide to a nucleic acid encoding a known polypeptide.The particular signal will depend on the label used in the relevantassay, e.g., a fluorescent label, a calorimetric label, a radioactivelabel, or the like. Labeled hybridization or PCR probes for detectingrelated polynucleotide sequences may be produced by oligolabeling, nicktranslation, end-labeling, or PCR amplification using a labelednucleotide.

A further indication that two nucleotide sequences are substantiallyidentical is that proteins encoded by the polynucleotides aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. Nucleic acid molecules thatdo not hybridize to teach other under stringent conditions are stillsubstantially identical if the corresponding proteins are substantiallyidentical. This may occur, for example, when two nucleotide sequencescomprise conservatively substituted variants as permitted by the geneticcode. Conservatively substituted variants refer to nucleotide sequenceshaving degenerate codon substitution wherein the third position of oneor more (or all) codons is/are substituted with mixed-base and/ordeoxyinosine residues. See Batzer et al., Nucleic Acids Res, 19:5081(1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1991); andRossolini et al., Mol. Cell. Probes 8:91-98 (1994).

Methods using manual alignment of sequences similar or homologous to oneor more polynucleotide sequences or one or more polypeptides encoded bythe polynucleotide sequences may be used to identify regions ofsimilarity and conserved domains. Such manual methods are well known topersons skilled in the art and can include, for example, comparisons ofthe tertiary structure between a polypeptide sequence encoded by apolynucleotide that comprises a known function with a polypeptidesequence encoded by a nucleotide sequence that has a function not yetdetermined. Examples of tertiary structure may include predicted alphahelices, beta-sheets, amphipathic helices, leucine zipper motifs, zincfinger motifs, proline-rich regions, cysteine repeat motifs, and thelike.

In embodiments of the aspects of the invention, the polynucleotides mayinclude polynucleotides encoding the WRKY76 transcription factorpolypeptide (protein) of SEQ ID NO: 2 or a variant thereof, such as aWRKY76 transcription factor polypeptide derived from the full-lengthamino acid sequence of SEQ ID NO: 2 containing one or moresubstitutions, deletions and/or additions of amino acid residues. Thepolynucleotides may therefore also include polynucleotides encoding afunctional variant WRKY76 polypeptide as described herein, for exampleSEQ ID NO: 12.

Orthologs and paralogs of transcription factor polypeptides describedherein may be cloned according to conventional methods. These may haveat least 80%, 85%, 90 or 95% sequence identity to HaWRKY76. For example,cDNAs can be cloned using mRNA from a plant cell or tissue thatexpresses one of the transcription factor polypeptides described herein.Appropriate mRNA sources may be identified by interrogating Northernblots with probes designed from amino acid sequences within the scope ofthe present disclosure, after which a library is prepared from the mRNAobtained from a positive cell or tissue. Transcription factor-encodingcDNA is then isolated by, for example, PCR, using primers designed froma transcription factor gene sequence disclosed herein, or by probingwith a partial or complete cDNA or with one or more sets of degenerateprobes based on sequences disclosed herein. The cDNA library may be usedto transform plant cells, as discussed further below, and expression ofthe cDNAs of interest is detected using, for example, methods known ordescribed herein, such as microarrays, Northern blots, quantitative PCR,or any other technique for monitoring changes in expression. Genomicclones may also be isolated using similar techniques.

In embodiments of the aspects of the invention, the polynucleotides maybe cloned, synthesized, altered, mutagenized, or combinations thereof. Anucleic acid can be isolated using standard molecular biology techniquesand the sequence information provided herein. Standard recombinant DNAand molecular cloning techniques used to isolate nucleic acids are knownto persons skilled in the art. In embodiments, a nucleic acid moleculecan be amplified using cDNA or, alternatively, genomic DNA, as atemplate and appropriate oligonucleotide primers according to standardPCT amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis.

WRKY76 Transcription Factor Polypeptides (Proteins)

Embodiments according to the various aspects of the invention alsorelate to isolated WRKY76 transcription factor polypeptides. The termpolypeptides (proteins) refers to compounds made up of a single chain ofamino acids joined by peptide bonds.

Representative polypeptides according to embodiments of the variousaspects of the invention include the full-length amino acid sequence ofSEQ ID NO: 2, and variants thereof, including substantially identicalpolypeptides. One variant is shown in SEQ ID NO: 12 Substantiallyidentical polypeptides have amino acid sequences that vary from thefull-length amino acid sequence of SEQ ID NO: 2 by one or moredeletions, substitutions, or additions, the net of which is retainedbiological function of the WRKY76 polypeptide. In embodiments, theWRKY76 polypeptides have at least 80% sequence identity with thefull-length amino acid sequence of SEQ ID NO: 2 as described herein, orby visual inspection. In embodiments, the WRKY76 polypeptides have atleast 85% sequence identity, or at least 90% sequence identity, or atleast 91% sequence identity, or at least 92% sequence identity, or atleast 93% sequence identity, or at least 94% sequence identity, or atleast 95% sequence identity, or at least 96% sequence identity, or atleast 97% sequence identity, or at least 98% sequence identity, or atleast 99% sequence identity with the full-length amino acid sequence ofSEQ ID NO: 2.

In some embodiments, substantially identical polypeptides contain atleast one of the following: (a) proline as the amino acid correspondingto position 270 of the full-length amino acid sequence of SEQ ID NO: 2or a variant thereof, for example SEQ ID NO: 12; (b) proline as theamino acid corresponding to position 260 of the full-length amino acidsequence of SEQ ID NO: 2 or a variant thereof, for example SEQ ID NO:12; (c) proline as the amino acid corresponding to position 87 of thefull-length amino acid sequence of SEQ ID NO: 2 or a variant thereof,for example SEQ ID NO: 12; (d) serine as the amino acid corresponding toposition 123 of the full-length amino acid sequence of SEQ ID NO: 2 or avariant thereof, for example SEQ ID NO: 12; (e) leucine as the aminoacid corresponding to position 14 of the full-length amino acid sequenceof SEQ ID NO: 2 or a variant thereof, for example SEQ ID NO: 12; (f)leucine as the amino acid corresponding to position 22 of thefull-length amino acid sequence of SEQ ID NO: 2 or a variant thereof,for example SEQ ID NO: 12; and (g) serine as the amino acidcorresponding to position 23 of the full-length amino acid sequence ofSEQ ID NO: 2 or a variant thereof, for example SEQ ID NO: 12. Inparticular, in one aspect, the invention relates to an isolatedpolypeptide comprising a sequence having at least 80% sequence identitywith the full-length amino acid sequence of SEQ ID NO: 2, thepolypeptide sequence containing at least one of:

-   -   (a) proline as the amino acid corresponding to position 270 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12;    -   (b) proline as the amino acid corresponding to position 87 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12;    -   (c) proline as the amino acid corresponding to position 260 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12;    -   (d) serine as the amino acid corresponding to position 123 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12;    -   (e) leucine as the amino acid corresponding to position 14 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12;    -   (f) leucine as the amino acid corresponding to position 22 of        the full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12; and    -   (g) serine as the amino acid corresponding to position 23 of the        full-length amino acid sequence of SEQ ID NO: 2 or a variant        thereof, for example SEQ ID NO: 12.

Substantially identical sequences according to the embodiments of thevarious aspects of the invention may be polymorphic sequences, i.e.,alternative sequences or alleles in a population in which the allelicdifference may be as small as one base pair. Substantially identicalpolynucleotides may also comprise mutagenized sequences, includingsequences comprising silent mutations. A mutation may comprise one ormore residue changes, a deletion of one or more residues, or aninsertion of one or more additional residues. In some embodiments,polypeptide variants can be functional fragments of the WRKY76transcription factor polypeptide of SEQ ID NO: 2. Functionalpolypeptides may include amino acid sequences that are longer than thesequences described herein. For example, one or more amino acids may beadded to the N-terminus or C-terminus of a polypeptide. Such additionalamino acids may be employed in a variety of applications, including (butnot limited to) purification applications. Methods of preparingelongated polypeptides are known to persons skilled in the art. In oneembodiment, the isolated polypeptide comprises or consists of SEQ IDNO:2 or 12.

WRKY76 transcription factor polypeptides described herein and accordingto the various aspects of the invention may comprise naturally occurringamino acids, synthetic amino acids, genetically encoded amino acids,non-genetically encoded amino acids, and combinations thereof.

WRKY76 polypeptides described herein and according to the variousaspects of the invention may also include polypeptides comprising aminoacids that are conservatively substituted variants of the full-lengthamino acid sequence of SEQ ID NO: 2. A conservatively substitutedvariant refers to a polypeptide comprising an amino acid in which one ormore residues have been conservatively substituted with a functionallysimilar residue. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue (e.g., isoleucine,valine, leucine, or methionine) for another, such as between arginineand lysine, between glutamine and asparagine, between glycine andserine; the substitution of one basic residue (e.g., lysine, arginine,or histidine) for another; or the substitution of one acidic residue(e.g., aspartic acid or glutamic acid) for another.

Isolated polypeptides according to embodiments may be purified andcharacterized using a variety of standard techniques that are known topersons skilled in the art. See Schroder et al., The Peptides, AcademicPress, New York, N.Y. (1965).

Regulatory Elements

The invention also relates to a vector or nucleic acid constructcomprising a polynucleotide as described above. In one embodiment, saidisolated polynucleotide comprises or consists of SEQ ID NO: 1 or 11. Inanother aspect, the invention relates to a recombinant expressioncassette comprising a polynucleotide as described above, wherein thepolynucleotide is operably linked to a promoter. The polynucleotide maybe in a sense or antisense orientation. In one embodiment, said isolatedpolynucleotide comprises or consists of SEQ ID NO: 1. In another aspect,the invention relates to recombinant expression cassette comprising anisolated polynucleotide operably linked to a promoter, wherein thepolynucleotide is a member selected from the group consisting of:

-   -   (a) a polynucleotide that encodes the polypeptide of SEQ ID NO:        2 or 12; and    -   (b) the polynucleotide of SEQ ID NO: 1 or a variant thereof, for        example SEQ ID NO: 11.

A regulatory element generally can increase or decrease the amount oftranscription of a nucleotide sequence operatively linked to the elementwith respect to the level at which the nucleotide sequence would betranscribed absent the regulatory element.

In some embodiments of the aspects of the invention, stress-regulatedregulatory elements, which regulate expression of an operatively linkednucleotide sequence in a plant in response to a stress condition, arealso provided. The plant stress-regulated regulatory elements may beisolated from a polynucleotide sequence of a plant stress-regulatedgene. Specifically, the plant stress-regulated regulatory elements maybe isolated from a polynucleotide sequence of the WRKY76 gene comprisingthe nucleotide sequence of SEQ ID NO: 1, or a variant thereof orcomprising a nucleotide sequence that is functionally equivalent to thefull-length nucleotide sequence of SEQ ID NO: 1, for example SEQ ID NO:11. Thus, a WRKY76 promoter, for example the HaWRKY76 promoter, may beused. This can be selected from SEQ ID NO: 10 or a sequence with atleast 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 10.

Methods for identifying and isolating a stress-regulated regulatoryelement from the polynucleotides, or genomic DNA clones correspondingthereto, are known to persons skilled in the art. For example, methodsof making deletion constructs or linker-scanner constructs can be usedto identify nucleotide sequences that are responsive to a stresscondition. Generally, such constructs include a reporter geneoperatively linked to the sequence to be examined for regulatoryactivity. By performing such assays, a plant stress-regulated regulatoryelement can be defined within a sequence of about 500 nucleotides orfewer, generally at least about 200 nucleotides or fewer, or about 50 to100 nucleotides. Preferably, the minimal (core) sequence required forregulating a stress response of a plant is identified. The nucleotidesequences of the genes of a cluster can also be examined using ahomology search engine to identify sequences of conserved identity,particularly in the nucleotide sequence upstream of the transcriptionstart site.

Regulatory elements, as described and defined herein, may be isolatedfrom a naturally occurring genomic DNA sequence or can be synthetic(e.g., a synthetic promoter). The regulatory elements can beconstitutively expressed so as to maintain gene expression at a relativelevel of activity (basal level), or can be regulated. Constitutivelyexpressed regulatory elements can be expressed in any cell type, or canbe tissue specific (expressed only in particular cell types), or phasespecific (expressed only during particular developmental or growthstages of a plant cell). Regulatory elements (e.g., a tissue specific,phase specific, or inducible regulatory element) useful in constructinga recombinant polynucleotide or in practicing methods described hereininclude regulatory elements that are found in a plant genome. In someembodiments, the regulatory elements may be from an organism other thana plant, such as a plant or animal virus, or an animal or othermulticellular organism.

In some embodiments, a regulatory element that is a promoter element isprovided. Particularly useful promoters include, but are not limited to,constitutive, inducible, temporally regulated, developmentallyregulated, spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences aregenerally understood to be strong or weak. A strong promoter providesfor a high level of gene expression, whereas a weak promoter providesfor a low level of gene expression. An inducible promoter is a promoterthat allows gene expression to be turned on and off in response to anexogenously added agent, or to an environmental or developmentalstimulus. An isolated promoter sequence that is a strong promoter forheterologous nucleic acid is typically advantageous because it providesfor a sufficient level of gene expression to allow for easy detectionand selection of transformed cells, while providing a high level of geneexpression when desired.

Several domains within a plant promoter region are necessary for thefull function of the promoter. The first of these domains within thepromoter region lies immediately upstream of the structural gene andforms the “core promoter region” containing consensus sequences,normally 70 base pairs immediately upstream of the gene. The corepromoter region represents a transcription initiation sequence thatdefines the transcription start point for the structural gene. Thepresence of the core promoter region defines a sequence as being apromoter; that is, if the region is absent, the promoter isnon-functional. The core promoter region on its own is, however,insufficient to provide full promoter activity. A series of regulatorysequences upstream of the core constitute the remainder of the promoter.These regulatory sequences determine expression levels, the spatial andtemporal patterns of expression and, for the specific subset ofpromoters, the expression level under inductive conditions (e.g., light,temperature, chemicals, hormones).

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′-region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques known to persons skilled in the art. Thereporter gene is operably linked downstream of the promoter, so thattranscripts initiating at the promoter proceed through the reportergene. Reporter genes generally encode proteins that are easily measured.The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate plant cell bytransfection techniques known to persons skilled in the art. The levelof enzyme activity corresponds to the amount of enzyme produced, which,in turn, reveals the level of expression from the promoter of interest.This level of expression can be compared to that achieved using otherpromoters to determine the relative strength of the promoter understudy. To ensure that the expression level is due to the promoter,rather than the stability of the mRNA, the level of the reporter mRNAcan be measured directly (e.g., by Northern blot analysis).

Once enzyme activity is detected, mutational and/or deletional analysesmay be performed to determine the minimal region and/or sequencesrequired to initiate transcription. Sequences may be deleted at the5′-end of the promoter region and/or at the 3′-end of the promoterregion, and nucleotide substitutions may be introduced. These constructsmay then be introduced into cells and their activity determined.

The promoter selection depends on the temporal and spatial requirementsfor expression, as well as on the target species. In some embodiments,expression in multiple tissues may be desirable, while in others,tissue-specific (e.g., leaf-specific, seed-specific, petal-specific,anther-specific, or pith-specific) expression is desirable. Althoughpromoters from dicotyledons have been shown to be operational inmonocotyledons, and vice versa, dicotyledonous promoters are ideallyselected for expression in dicotyledons, and monocotyledonous promotersare ideally selected for expression in monocotyledons. There is norestriction as to the origin or source of the promoter selected; it issufficient that the selected promoter is operational in driving theexpression of a nucleotide sequence described herein in the particularcell. That is, the promoter used in embodiments of the presentdisclosure may be any nucleotide sequence that shows transcriptionalactivity in the target (host) plant (cell, seed, etc.).

Accordingly, in embodiments, the promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequencedisclosed herein. Where the promoter is native or endogenous to theplant host, it is intended that the promoter is found in the nativeplant into which the promoter is introduced. Where the promoter isforeign or heterologous to the DNA sequence disclosed herein, thepromoter is not the native or naturally occurring promoter for theoperably linked DNA sequence disclosed herein. The promoter selected inembodiments may be “inducible” or “constitutive.” An inducible promoteris a promoter that is under environmental control, whereas aconstitutive promoter is a promoter that is active under mostenvironmental conditions. Moreover, the promoter may benaturally-occurring, composed of portions of various naturally-occurringpromoters, or partially or totally synthetic. Guidance for the design ofpromoters is provided by studies of promoter structure in Harley et al.,Nucleic Acids Res. 15:2343-61 (1987). Additionally, the location of thepromoter relative to the transcription start position may be optimized.See e.g., Roberts et al., Proc. Natl. Acad. Sci. 76:760-764, USA (1979).

For example, suitable constitutive promoters for use in plants accordingto the present disclosure may include promoters from plant viruses, suchas the peanut chlorotic streak caulimovirus (PC1SV) promoter, the 35Spromoter from cauliflower mosaic virus (CaMV), promoters of Chlorellavirus methyltransferase genes, the full-length transcript promoter fromfigwort mosaic virus (FMV); the promoters from such genes as rice actin,ubiquitin, pEMU, MAS, maize H4 histone, Brassica napus ALS4; andpromoters of various Agrobacterium genes. See e.g., Odell et al., Nature313:810-812 (1985); McElroy et al., Plant Cell 2:163-171 (1990);Christensen et al., Plant Mol. Biol. 12:619-632 (1989); Christensen etal., Plant Mol. Biol. 18:675-689 (1992); Last et al., Theor. Appl.Genet. 81:581-588 (1991); Velten et al., EMBO J. 3:2723-27310 (1984);Lepetit et al., Mol. Gen. Genet. 231:276-285 (1992); and U.S. Pat. Nos.4,771,002; 5,102,796; 5,182,200; 5,428,147; 5,850,019; 5,563,328;5,378,619.

Suitable inducible promoters for use in plants according to the presentdisclosure may include, for example, the promoter from the ACE1 systemthat responds to copper, the promoter of the maize In2 gene thatresponds to benzenesulfonamide herbicide safeners, and the promoter ofthe Tet repressor from Tn10. See Mett et al., Proc. Natl. Acad. Sci.,90:4567-4571, USA (1993); Hershey et al., Mol. Gen. Genet. 227:229-237(1991); and Gatz et al., Mol. Gen. Genet. 243:32-38 (1994). Anotherinducible promoter that may be used in plants described herein is onethat responds to an inducting agent to which plants do not normallyrespond. An inducible promoter of this type may be the induciblepromoter from a steroid hormone gene, the transcriptional activity ofwhich is induced by glucocorticosteroid hormone, or the recentapplication of a chimeric transcription activator, SVE, for use in anestrogen receptor-based inducible plant expression system activated byestradiol. See Schena et al., Proc. Natl. Acad. Sci. 88:104-21 (1991);Zuo et al., Plant J. 24:265-273 (2000). Other inducible promoterssuitable for use in embodiments may be selected from promoters describedin EP 332104, PCT International Publication Nos. WO93/21334 and WO97/06269. Promoters composed of portions of other promoters andpartially or totally synthetic promoters may also be used. See e.g., Niet al., Plant J. 7:661-676 (1995); and PCT International Publication No.95/14098 (describing use of such promoters in plants).

In embodiments, the promoter may be a WRKY76-specific promoter clonedaccording to methods described herein.

The promoter may include, or be modified to include, one or moreenhancer elements to thereby provide for higher levels of transcription.Examples of suitable enhancer elements for use in plants describedherein include, for example, the PC1SV enhancer element, the CaMV 35Senhancer element and the FMV enhancer element. See Maiti et al.,Transgenic Res. 6:143-156 (1997); PCT International Publication No. WO96/23898; and U.S. Pat. Nos. 5,850,019; 5,106,739; and 5,164,316.

Expression Constructs

Embodiments include recombinant constructs comprising one or more of thepolynucleotide sequences described herein. The constructs typicallycomprise a vector, such as a plasmid, a cosmid, a phage, a virus, abacterial artificial chromosome (BAC), a yeast artificial chromosome(YAC), or the like, into which a polynucleotide sequence as describedherein has been inserted, in a forward or reverse orientation. In someembodiments, the constructs may further comprise regulatory sequences,including, e.g., a promoter that is operably linked to the sequence.Vectors and promoters suitable for recombinant constructs of the presentdisclosure may include those generally known to persons having skill inthe art and/or described herein.

Constructs suitable for use in embodiments may contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the polypeptide of interest to certainintracellular structures, such as the chloroplast (or other plastid),endoplasmic reticulum, or Golgi apparatus, or to be secreted. Suchsequences include leader sequences targeting transport and/orglycosylation by passage into the endoplasmic reticulum, vacuoles,plastids including chloroplasts, mitochondria, and the like. Forexample, the constructs may be engineered to contain a signal peptide tofacilitate transfer of the peptide to the endoplasmic reticulum. Asignal sequence is known or suspected to result in co-translational orpost-translational peptide transport across the cell membrane. Ineukaryotes, this typically involves secretion into the Golgi apparatus,with some resulting glycosylation. Leader sequence refers to anysequence that, when translated, results in an amino acid sequencesufficient to trigger co-translational transport of the peptide chain toa sub-cellular organelle. Plant expression cassettes may also contain anintron, such that mRNA processing of the intron is required forexpression

Suitable constructs may also contain 5′ and 3′ untranslated regions. A3′ untranslated region is a polynucleotide located downstream of acoding sequence. Polyadenylation signal sequences and other sequencesencoding regulatory signals capable of affecting the addition ofpolyadenylic acid tracts to the 3′ end of the mRNA precursor or the 3′untranslated regions. A 5′ untranslated region is a polynucleotidelocated upstream of a coding sequence.

Any of the sequences described herein may be incorporated into acassette or vector for expression in plants. Expression vectors suitablefor stable transformation of plant cells or for the establishment oftransgenic plants are known by persons skilled in the art. See e.g.,Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, (1989); and Gelvin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers (1990). Specific examples may include those derivedfrom a Ti plasmid of Agrobacterium tumefaciens and those described foruse in dicotyledonous plants in Herrera-Estrella et al., Nature 303:209(1983) and Klee, Bio/Technol. 3:637-642 (1985). In embodiments, non-Tivectors may be used to transfer the DNA into monocotyledonous plants andcells by using free DNA delivery techniques. Such methods may involve,for example, the use of liposomes, electroporation, microprojectilebombardment, silicon carbide whispers, and viruses.

The termination region may be native to the transcriptional initiationregion, the sequence described herein, or may be derived from anothersource. Suitable termination regions may be derived from the Ti-plasmidof A. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions, or the termination region of a plant gene, such assoybean storage protein. See Guerineau et al., Mol. Gen. Genet.262:141-144 (1991); Proudfoot, Cell 64:671-674 (1991); Sanfacon et al.,Genes Dev. 5:141-149 (1991); Mogen et al., Plant Cell 2:1261-1272(1990); Munroe et al., Gene 91:151-158 (1990); Ballas et al., NucleicAcids Res. 17:7891-7903 (1989); and Joshi et al., Nucleic Acids Res.15:9627-9639 (1987). These vectors are plant integrating vectors in thatupon transformation, the vectors integrate a portion of vector DNA intothe genome of the host plant. An example of a vector useful inembodiments is plasmid pBI121.

Where appropriate, the vector and WRKY76 transcription factor sequencesdisclosed herein may be optimized for increased expression in thetransformed host cell. That is, the sequences may be synthesized usinghost cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the polynucleotide will be increased. Seee.g., Campbell et al., Plant Physiol. 92:1-11 (1990). Methods forsynthesizing host-preferred polynucleotides are known by persons skilledin the art. See e.g., Murray et al., Nucleic Acids Res. 17:477-498(1989); U.S. Patent Application Publications Nos. 2004/0005600 and2001/0003849; and U.S. Pat. Nos. 6,320,100; 6,075,185; 5,380,831; and5,436,391, the entire disclosures of which are incorporated by referenceherein.

For example, polynucleotides of interest can be targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, an expressioncassette according to some embodiments may additionally contain apolynucleotide encoding a transcription factor polypeptide to direct thenucleotide of interest to the chloroplasts. Such transit peptides areknown by persons skilled in the art. See e.g., Von Heijne et al., PlantMol. Biol. Rep. 9:104-126 (1991); Clark et al., J. Biol. Chem.264:17544-17550 (1989); Della-Cioppa et al., Plant Phsyiol. 84:965-968(1987); Romer et al., Biochem. Biophys. Res. Commun. 196:1414-1421(1993); and Shah et al., Science 233:478-481 (1986). The polynucleotidesof interest to be targeted to the chloroplast may further be optimizedfor expression in the chloroplast to account for differences in codonusage between the plant nucleus and this organelle. In this manner, thepolynucleotides of interest may be synthesized usingchloroplast-preferred codons. See U.S. Pat. No. 5,380,831, the entiredisclosure of which is incorporated by reference herein.

In embodiments, one or more plant expression cassette (i.e., a WRKY76transcription factor open reading frame operably linked to a promoter)may be inserted into a plant transformation vector, which allows for thetransformation of DNA into a cell. Such expression cassettes may beorganized into more than one vector DNA molecule.

Plant expression vectors suitable for use in embodiments may compriseone or more DNA vector(s) for achieving plant transformation. Forexample, it is common practice for persons skilled in the art to utilizeplant transformation vectors that include one or more cloned plantcoding sequences (genomic or cDNA) under the transcriptional control of5′ and 3′ regulatory sequences as a dominant selectable marker. Suchplant transformation vectors typically also contain a promoter, atranscription initiation start site, an RNA processing signal, atranscription termination site, and/or a polyadenylation signal.

Binary vectors are plant transformation vectors that utilize twonon-contiguous DNA vectors to encode all requisite cis- and trans-actingfunctions for transformation of plant cells. See Hellens et al., Trendsin Plant Sicence 5:446-451 (2000). Binary vectors, as well as vectorswith helper plasmids, are most often used for Agrobacterium-mediatedtransformations, in which the size and complexity of DNA segments neededto achieve efficient transformation is large, and in which it istherefore advantageous to separate functions among separate DNAmolecules. Binary vectors also typically contain a plasmid vector thatcontains the cis-acting sequence required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a polynucleotide ofinterest (i.e., a polynucleotide engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Sequences required for bacterial replication may also be present on thisplasmid vector. The cis-acting sequences are arranged in a fashion toallow for efficient transfer into plant cells and expression therein.For example, a selectable marker sequence and a sequence of interest aretypically located between the left and right borders. Often a secondplasmid vectors the trans-acting factors that mediate T-DNA transferfrom Agrobacterium to the target (host) plant cells. This plasmidtypically contains virulence functions (Vir genes) that allow infectionof plant cells by Agrobacterium, and transfer of DNA by cleavage atborder sequences and vir-mediated DNA transfer, as understood by personsskilled in the art. See e.g., Hellens et al., Trends in Plant Science5:446-451 (2000). Several types of Agrobacterium strains (e.g., LBA4404,GV3101, EHA101, EHA105, etc.) may be used for plant transformation. Thesecond plasmid vector is typically not necessary for introduction ofpolynucleotides into plants by other methods, such as bymicroprojection, microinjection, electroporation, etc.

In some embodiments, expression vectors include RNA processing signalsthat can be positioned within, upstream or downstream of the codingsequence. The expression vectors may include additional regulatorysequences from the 3′-untranslated region of plant genes. Initiationsignals may also be used to aid in efficient translation of codingsequences. These signals can include, e.g., an ATG initiation codon andadjacent sequences. In cases where a coding sequence, its initiationcodon and upstream sequences are inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only coding sequences, or a portionthereof, are inserted, exogenous transcriptional control signalsincluding the ATDG initiation codon can be separately provided. Theinitiation codon is provided in the correct reading frame to facilitatetranscription. Exogenous transcription elements and initiation codonscan be of various origins, both natural and synthetic. The efficiency ofexpression can be enhanced by including enhancers appropriate for thecell system in use.

In embodiments where co-suppression of a gene is desired, vectors inwhich RNA encoded by a transcription factor or transcription factorhomologue cDNA is over-expressed may be used to obtain co-suppression ofa corresponding endogenous gene. See e.g., U.S. Pat. No. 5,231,020. Suchco-suppression (also termed “sense suppression”) does not require thatthe entire transcription factor cDNA be introduced into the plant cells,nor does it require that the introduced sequence is identical to theendogenous transcription factor gene of interest. However, as withantisense suppression, the suppressive efficiency will be enhanced asthe specificity of hybridization is increased.

Embodiments include host (i.e., target) cells transduced with vectorsdescribed herein, and the production of polypeptides by recombinanttechniques. Host cells are genetically engineered (i.e., nucleic acidsare introduced by transduction, transformation or transfection) with thevectors, which may be, e.g., a cloning vector or an expression vectorcomprising the relevant nucleic acids described herein. The vector mayoptionally be, for example, a plasmid, a viral particle, a phage, anaked nucleic acid, etc.

Using polynucleotides disclosed herein, a protein may be expressed in arecombinantly engineered cell, such as a plant cell. Persons skilled inthe art are knowledgeable about various expression systems available forexpression of a polynucleotide encoding a protein according toembodiments described herein.

The expression of isolated polynucleotides encoding a protein accordingto embodiments described herein will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter, followed byincorporation thereof into an expression vector. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein described herein. To obtain a high level ofexpression of a cloned gene, it is typically desirable to constructexpression vectors that contain, at minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation termination site. Persons having ordinaryskill in the art would recognize that modifications could be made to aprotein of the present disclosure without diminishing its biologicalactivity.

Embodiments and aspects of the invention include an expression cassette.The expression cassette that comprises at least: (1) a constitutive,inducible, or tissue-specific promoter; and (2) a recombinantpolynucleotide having a polynucleotide sequence, or a complementarypolynucleotide sequence thereof, selected from the group consisting of apolynucleotide sequence encoding: (a) a polypeptide sequence having atranscription factor sequence as described herein; (b) a polynucleotidesequence selected from the transcription factor polynucleotidesdescribed herein; or sequence variants (e.g., allelic or splicevariants) of the polynucleotide sequences referenced in (a) or (b)above, where the sequence variant encodes a polypeptide that regulatestranscription.

Expression Hosts

The invention also relates to host cells comprising polynucleotides asdescribed above, nucleic acid constructs, vectors or expressioncassettes as described above. For example, the polynucleotide comprisesor consists of SEQ ID NO:1 or a variant thereof, or SEQ ID NO:9 or avariant thereof. In one embodiment, the variant comprises or consists ofSEQ ID NO:11.

In some embodiments, host cells are transduced with vectors as describedherein. Host cells are genetically engineered (i.e., nucleic acids areintroduced, transformed or transfected) with the vectors describedherein, which may be, e.g., a cloning vector or an expression vectorcomprising the relevant nucleic acids disclosed herein. The vector isoptionally a plasmid, a viral particle, a phage, a nucleic acid, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the relevant gene. The culture conditions,such as temperature, pH and the like, may be those previously used withthe host cell selected for expression, and will be apparent to thoseskilled in the art.

The host cell may be a eukaryotic cell, such as a yeast cell or a plantcell, or the host cell may be a prokaryotic cell, such as a bacterialcell, for example Agrobacterium. In some embodiments, plant protoplastsmay be suitable for use. In preferred embodiments, the host cell is aplant cell. A kit comprising such a host cell is also within the scopeof the invention.

For example, the DNA fragments may be introduced into plant tissues,cultured plant cells or plant protoplasts by standard methods,including, e.g., electroporation, infection by viral vectors such ascauliflower mosaic virus (CaMV), high velocity ballistic penetration bysmall particles with the nucleic acid either within the matrix of smallbeads or particles, or on the surface, use of pollen as a vector, orusing Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNAplasmid in which DNA fragments are cloned. See Fromm et al., Proc. Natl.Acad. Sci. 82:8524-5828 (1985); Hohn et al., Molecular Biology of PlantTumors, Academic Press, New York, N.Y. pp. 549-560 (1982); U.S. Pat. No.4,407,956; Klein et al., Nature 327:70-73 (1987); PCT InternationalPublication No. WO 85/01856. The T-DNA plasmid is transmitted to plantcells upon infection by Agrobacterium tumefaciens, and a portion isstably integrated into the plant genome. See Horsch et al., Science233:496-498 (1984); and Fraley et al., Proc. Natl. Acad. Sci.80:4803-4807 (1983).

The host cell may include a nucleic acid as described above. In someembodiments the host cell may include a nucleic acid that encodes apolypeptide according to embodiments described above, such that the cellexpresses a polypeptide of interest as described herein. In someembodiments, the cell may include vector sequences or the like. As willbe understood by persons skilled in the art, cells and transgenic plantsthat include any polypeptide or polynucleotide sequence described herein(e.g., produced by transduction of a vector described herein) representembodiments within the scope of the present disclosure.

For long-term, high-yield production of recombinant proteins, stableexpression may be used. Host cells transformed with a nucleotidesequence encoding a polypeptide as disclosed herein are optionallycultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein or fragment thereofproduced by a recombinant cell may be secreted, membrane-bound, orcontained intracellularly, depending on the sequence and/or the vectorused. As will be understood by persons skilled in the art, expressionvectors according to embodiments containing polynucleotides that encodemature proteins can be designed with signal sequences that directsecretion of the mature polypeptides through a prokaryotic or eukaryoticcell membrane.

Some embodiments of the invention relate to recombinant expression of aWRKY76 transcription factor protein in a stable cell line. Methods forgenerating a stable cell line following transformation of a heterologousconstruct into a host cell are known in the art. The transformed cells,tissues, and plants are therefore understood to encompass not only theend product of a transformation process, but also transgenic progeny orpropagated forms thereof.

Transgenic Plants and Production Thereof

Embodiments also relate to transgenic plants, methods of producing thetransgenic plants, and methods for modifying plant traits or conferringdesirable traits upon host plants to produce transgenic plants havingimproved stress tolerance and yield in standard growing conditions. Alsowithin the scope of the invention are plants obtained or obtainable bythe methods described herein.

Polynucleotides disclosed herein are favorably employed to producetransgenic plants with various traits or characteristics that have beenmodified in a desirable manner, e.g., to improve the seedcharacteristics of the plant. For example, altering the expressionlevels or patterns of one or more of the transcription factors (ortranscription factor homologues) disclosed herein, as compared with thelevels of the same protein found in a control wild-type plant, can beused to modify a plant's traits. Illustrative examples of traitmodification and improved characteristics resulting from alteringexpression levels of the disclosed WRKY76 transcription factor sequencesare explained in more detail in the various Examples below.

In some aspects of the invention, plants expressing a heterologousWRKY76 transcription factor, including plants that express a WRKY76transcription factor at elevated levels, are provided. Still otheraspects of the invention relate to the generation of plants withconditional or inducible expression of a WKKY transcription factorprotein as disclosed herein.

Thus, in one further aspect, the invention relates to a transgenic plantcomprising and expressing a nucleic acid construct, vector or expressioncassette comprising a nucleic acid that encodes a WRKY76 transcriptionfactor. For example, said nucleic acid comprises or consists of SEQ IDNO:1 or a variant thereof, for example SEQ ID NO:11, or SEQ ID NO:9, afunctional variant or part thereof as defined above. In one embodiment,said polynucleotide has at least 80% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1 or a variant thereof,for example SEQ ID NO:11. In another embodiment, said polynucleotide hasat least 80% sequence identity with the full-length nucleotide sequenceof SEQ ID NO: 1 or a variant thereof, for example SEQ ID NO:11, whereinthe polynucleotide contains at least one of:

-   -   (a) adenine as the nucleotide corresponding to position 817 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (b) cytosine as the nucleotide corresponding to position 808 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (c) cytosine as the nucleotide corresponding to position 33 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (d) cytosine as the nucleotide corresponding to position 259 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11; and    -   (e) guanine as the nucleotide corresponding to position 315 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11.

A plant according to the various aspects of the invention, including thetransgenic plants, methods and uses described herein may be a monocot ora dicot plant. A dicot plant may be selected from the familiesincluding, but not limited to Asteraceae, Brassicaceae (e.g. Brassicanapus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae,Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae,Rosaceae or Solanaceae. For example, the plant may be selected fromlettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash,cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple,rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea,lentil, peanut, chickpea, apricots, pears, peach, grape vine, bellpepper, chilli or citrus species. A monocot plant may, for example, beselected from the families Arecaceae, Amaryllidaceae or Poaceae. Forexample, the plant may be a cereal crop, such as maize, wheat, rice,barley, oat, sorghum, rye, millet, buckwheat, or a grass crop such asLolium species or Festuca species, or a crop such as sugar cane, onion,leek, yam or banana. Also included are biofuel and bioenergy crops suchas rape/canola, sugar cane, sweet sorghum, Panicum virgatum(switchgrass), linseed, lupin and willow, poplar, poplar hybrids,Miscanthus or gymnosperms, such as loblolly pine. Also included arecrops for silage (maize), grazing or fodder (grasses, clover, sanfoin,alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine,oak), pulping (e.g. poplar), feeder stocks for the chemical industry(e.g. high erucic acid oil seed rape, linseed) and for amenity purposes(e.g. turf grasses for golf courses), ornamentals for public and privategardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) andplants and cut flowers for the home (African violets, Begonias,chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubberplant). Preferably, the plant is a crop plant. By crop plant is meantany plant which is grown on a commercial scale for human or animalconsumption or use. In a preferred embodiment, the plant is a cereal.Most preferred plants are maize, rice, wheat, oilseed rape/canola,sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape,barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet,broccoli or other vegetable brassicas or poplar.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, fruit, shoots,stems, leaves, roots (including tubers), flowers, tissues and organs,wherein each of the aforementioned comprise the nucleic acid constructas described herein. The term “plant” also encompasses plant cells,suspension cultures, callus tissue, embryos, meristematic regions,gametophytes, sporophytes, pollen and microspores, again wherein each ofthe aforementioned comprises the nucleic acid construct as describedherein.

The aspects of the invention also extend to products derived, preferablydirectly derived, from a harvestable part of such a plant, such as drypellets or powders, oil, fat and fatty acids, starch or proteins. Theinvention also relates to food products and food supplements comprisingthe plant of the invention or parts thereof.

The transgenic plants show increased tolerance to stress conditions, forexample abiotic and biotic stress compared to a control plant, forexample a wild type plant. In particular, the plants show increasedstress response to abiotic stress selected from drought or irrigation.

Plants expressing a heterologous WRKY76 transcription factor protein maybe further modified at more than one WRKY76 transcription factor locusor at a locus other than a WRKY76 transcription factor locus to conferincreased stress tolerance or another trait of interest.

The invention also relates to method for producing transgenic plantscomprising introducing and expressing in a plant a polynucleotide,vector or expression cassette as described above or a polynucleotideencoding a WRKY76 polypeptide as described above. Such methods comprisegenerating from the plant cell a transgenic plant that expresses thepolynucleotide. In one embodiment, the method comprises introducing andexpressing a nucleic acid that comprises or consists of SEQ ID NO:1, afunctional variant or part thereof as defined above. In one embodiment,said polynucleotide has at least 80% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1. In another embodiment,said polynucleotide has at least 80% sequence identity with thefull-length nucleotide sequence of SEQ ID NO: 1 or a variant thereof,for example SEQ ID NO:11 or SEQ ID NO:9, wherein the polynucleotidecontains at least one of:

-   -   (a) adenine as the nucleotide corresponding to position 817 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (b) cytosine as the nucleotide corresponding to position 808 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (c) cytosine as the nucleotide corresponding to position 33 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (d) cytosine as the nucleotide corresponding to position 259 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11; and    -   (e) guanine as the nucleotide corresponding to position 315 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11.

To prepare a plant expressing a heterologous WRKY76 transcription factoraccording to embodiments, the introduction of a WKKY polynucleotidedisclosed herein may be accomplished by techniques known in the art,including, but not limited to, electroporation or chemicaltransformation. See e.g., Ausubel, Current Protocols in MolecularBiology, John Wiley and Sons, Inc., Indianapolis, Ind. (1994). Markersconferring resistance to toxic substances may also be used to identifytransformed cells (having taken up and expressed the test polynucleotidesequence) from non-transformed cells (those not containing or notexpressing the test polynucleotide sequence). The stable transformationof “transformed” or “transgenic” plants as used herein refers to theintroduction of a polynucleotide construct into a plant, such that itintegrates into the genome of the plant and is capable of beinginherited by progeny thereof.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells, followed by applying a maximumthreshold level of appropriate selection to recover the transformedplant cells from an untransformed cell mass. Subsequently, thetransformed cells are differentiated into shoots after being placed on aregeneration medium supplemented with a maximum threshold level ofselecting agent (e.g., temperature, herbicide, etc.). The shoots arethen transferred to a selective rooting medium for recovering the rootedshoot or plantlet. The transgenic plantlet is then grown into a matureplant that produces fertile seeds. A general description of techniquesand methods for generating transgenic plants may be found in Ayres etal., CRC Crit. Rev. Plant Sci. 13:219-239 (1994), and Bommineni et al.,Maydica 42:107-120 (1997).

In general, since the transformed material contains many cells, bothtransformed and non-transformed cells are present in any piece ofsubjected target callus, tissue, or group of cells. The ability to killnon-transformed cells and allow transformed cells to proliferate resultsin transformed plant cultures. Often, the ability to removenon-transformed cells is a limitation to rapid recovery of transformedplant cells and successful generation of transgenic plants. Therefore,molecular and biochemical methods may be used for confirming thepresence of the integrated nucleotide(s) of interest in the genome ofthe transgenic plant. For example, selectable markers, such as enzymesresulting in a change of color or luminescent molecules (e.g., GUS andluciferase), antibiotic-resistant genes (e.g., gentamicin andkanamycin-resistance genes) and chemical-resistant genes (e.g.,herbicide-resistance genes) may be used to confirm the integration ofthe nucleotide(s) of interest in the genome of the transgenic plant.Alternatively, particularly in considering the safety of the transgenicplants, the transformed plants can be selected under environmentalstresses avoiding the incorporation of any selectable marker genes.

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells has become routine, and the selection of themost appropriate transformation technique can be readily determined bythe person skilled in the art. The choice of method will typically varybased on the type of plant to be transformed, as persons skilled in theart will recognize the suitability of particular methods for given planttypes. Suitable methods for use in embodiments may include, but are notlimited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium tumefaciens mediated transformation. Successful examplesof modifications of plant characteristics by transformation with clonedsequences, which serve to illustrate current knowledge in the art andare herein incorporated by reference, include: U.S. Pat. Nos. 5,571,706;5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871;5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369; and 5,610,042.

The generation of transgenic plants according to embodiments may beperformed by methods known to persons skilled in the art, including:introduction of heterologous DNA by Agrobacterium into plant cells(Agrobacterium-mediated transformation); bombardment of plant cells withheterologous foreign DNA adhered to particles; and various othernon-particle direct-mediated methods, such as microinjection,electroporation, application of Ti plasmid, Ri plasmid, or plant virusvector, and direct DNA transformation.

Generally, there are three types of conventional Agrobacterium-mediatedtransformation methods. The first method involves co-cultivation ofAgrobacterium with cultured isolated protoplasts. This method requiresan established culture system that allows culturing protoplasts andplant regeneration from cultured protoplasts. The second method involvestransformation of cells or tissues with Agrobacterium. This methodrequires that the plant cells or tissues can be transformed byAgrobacterium, and that the transformed cells or tissues can be inducedto regenerate into whole plants. The third method involves thetransformation of seeds, apices or meristems with Agrobacterium. Thismethod requires micropropagation.

The efficiency of Agrobacterium-mediated transformation methods may beenhanced by, e.g., including in the Agrobacterium culture a naturalwound response molecule, such as acetosyringone (AS), which has beenshown to enhance transformation efficiency with Agrobacteriumtumefaciens. See Shahla et al., Plant Molec. Biol. 8:291-298 (1987).Alternatively, transformation efficiency may be enhanced by wounding thetarget tissue to be transformed by, e.g., punching, maceration,bombardment with microprojectiles, etc. See e.g., Bidney et al., PlantMolec. Biol. 18:301-313 (1992).

In some embodiments, plant cells may be transfected with vectors viaparticle bombardment (e.g., with a gene gun). Particle mediated genetransfer methods are known in the art, are commercially available, andinclude, e.g., the gas driven gene delivery instrument described in U.S.Pat. No. 5,584,807, the contents of which are incorporated by referenceherein. This method involves coating the polynucleotide sequence ofinterest onto heavy metal particles, and accelerating the coatedparticles under the pressure of compressed gas for delivery to thetarget tissue.

In some embodiments, specific initiation signals may be used to achievemore efficient translation of sequences encoding a polypeptide describedherein, such as, e.g., the ATG initiation codon and adjacent sequences.In cases where sequences encoding the polypeptide of interest, itsinitiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However in cases where onlythe coding sequence or a portion thereof is inserted, heterologoustranslational control signals that include the ATG initiation codon maybe provided.

In addition to expression of nucleic acids disclosed herein as genereplacement or plant phenotype modification nucleic acids, disclosednucleic acids may also be used for sense and anti-sense suppression ofexpression, e.g., to down-regulate expression of the disclosed nucleicacids, as a further mechanism for modulating plant phenotypes. That is,nucleic acids described herein, or subsequences or anti-sense sequencesthereof, can be used to block expression of naturally occurringhomologous nucleic acids. A variety of sense and anti-sense technologiesis known in the art. See e.g., Lichtenstein and Nellen, AntisenseTechnology: A Practical Approach, IRL Press at Oxford University,Oxford, England (1997). In general, sense or anti-sense sequences areintroduced into a cell, where they are optionally amplified, e.g., bytranscription. Such sequences include both simple oligonucleotidesequences and catalytic sequences, such as ribozymes.

For example, the reduction or elimination of expression (i.e., a“knock-out”) of a transcription factor or transcription factor homologuepolypeptide in a transgenic plant, e.g., to modify a plant trait, can beobtained by introducing an antisense construct corresponding to thepolypeptide of interest as a cDNA. For antisense suppression, thetranscription factor or homologue cDNA is arranged in reverseorientation (with respect to the coding sequence) relative to thepromoter sequence in the expression vector. The introduced sequence neednot be the full length cDNA or gene, and need not be identical to thecDNA or gene found in the plant type to be transformed. Typically, theantisense sequence need only be capable of hybridizing to the targetgene or RNA or interest. Thus, where the introduced sequence is ofshorter length, a higher degree of homology to the endogenoustranscription factor sequence will be needed for effective antisensesuppression. While antisense sequences of various lengths can beutilized, preferably, the introduced antisense sequence in the vectorwill be at least 30 nucleotides in length, and improved antisensesuppression will typically be observed as the length of the antisensesequence increases. Preferably, the length of the antisense sequence inthe vector will be greater than 100 nucleotides. Transcription of anantisense construct as described herein results in the production of RNAmolecules that are the reverse complement of mRNA molecules transcribedform the endogenous transcription factor gene in the plant cell.

In some embodiments, suppression of endogenous transcription factor geneexpression can also be achieved using a ribozyme. Ribozymes are RNAmolecules that possess highly specific endoribonuclease activity. Theproduction and use of ribozymes are disclosed in U.S. Pat. Nos.4,987,071 and 5,543,508. Synthetic ribozyme sequences includingantisense RNAs can be used to confer RNA cleaving activity on theantisense RNA, such that endogenous mRNA molecules that hybridize to theantisense RNA are cleaved, which in turn leads to an enhanced antisenseinhibition of endogenous gene expression.

In some embodiments, vectors in which RNA encoded by a transcriptionfactor or transcription factor homologue cDNA is over-expressed may beused to obtain co-suppression of a corresponding endogenous gene, asdescribed in U.S. Pat. No. 5,231,020. Such co-suppression (also termed“sense suppression”) does not require that the entire transcriptionfactor cDNA to be introduced into the plant cell, nor does it requirethat the introduced sequence be exactly identical to the endogenoustranscription factor gene of interest. However, as with antisensesuppression, the suppressive efficiency will be enhanced as specificityof hybridization is increased, e.g., as the introduced sequence islengthened, and/or as the sequence similarity between the introducedsequence and the endogenous transcription factor gene is increased.

Vectors expressing an untranslatable form of the transcription factormRNA, e.g., sequences comprising one or more stop codon, or nonsensemutation, can also be used to suppress expression of an endogenoustranscription factor, thereby reducing or eliminating its activity andmodifying one or more traits. Methods for producing such constructs aredescribed in U.S. Pat. No. 5,583,021. Preferably, such constructs aremade by introducing a premature stop codon into the transcription factorgene.

Another method for abolishing the expression of a gene is by insertionmutagenesis using the T-DNA of Agrobacterium tumefaciens. Aftergenerating the insertion mutants, the mutants can be screened toidentify those containing the insertion in a transcription factor ortranscription factor homologue gene. Plants containing a singletransgene insertion even at the desired gene can be crossed to generatehomozygous plants for the mutation. See Koncz et al., Methods inArabidopsis Research, World Scientific (1992).

Alternatively, a plant phenotype can be altered by eliminating anendogenous gene, such as a transcription factor or transcription factorhomologue, e.g., by homologous recombination. See Kempin et al., Nature,389:820 (1997).

Polynucleotides and polypeptides disclosed herein can also be expressedin a plant in the absence of an expression cassette by manipulating theactivity or expression level of the endogenous gene by other means,e.g., by ectopically expressing a gene by T-DNA activation tagging. SeeIchikawa et al., Nature 390:698-701 (1997); and Kakimoto et al., Science274:982-985 (1996). This method entails transforming a plant with a genetag containing multiple transcriptional enhancers and, once the tag hasinserted into the genome, expression of a flanking gene coding sequencebecomes deregulated. As another example, the transcriptional machineryin a plant can be modified so as to increase transcription levels of apolynucleotide disclosed herein. See e.g., PCT InternationalPublications Nos. WO 96/06166 and WO 98/53057 (describing modificationsof DNA binding specificity of zinc finger proteins by changingparticular amino acids in the DNA binding motif).

Following transformation, the plants are preferably selected using adominant selectable marker incorporated into the transformation vector.After transformed plants are selected and grown to maturity, thoseplants showing a modified trait are identified. The modified train canbe any of the traits described herein. Additionally, to confirm that themodified trait is due to changes in expression levels or activity of thepolypeptide or polynucleotide disclosed herein, the mRNA expression maybe analyzed using Northern blots, RT-PCR or microarrays, or proteinexpression using immunoblots, Western blots, or gel shift assays.

In some embodiments, the plants may be homozygous for the WRKY76polynucleotide disclosed herein, i.e., a transgenic plant that containstwo added sequences, one sequence at the same locus on each chromosomeof a chromosome pair. A homozygous transgenic plant according to theseembodiments can be obtained by sexually mating (selfing) an independentsegregating transgenic plant that contains the added sequences disclosedherein, germinating some of the seed produced and analyzing theresulting plants produced for enhanced enzyme activity and/or increasedplant yield relative to a control (native, non-transgenic) or anindependent segregant transgenic plant. Persons skilled in the art willunderstand that two different transgenic plants may be mated to produceoffspring that contain two independently segregating added heterologouspolynucleotides.

Cells that have been transformed may be grown into plants inconventional ways. See e.g., McCormick et al., Plant Cell Rep. 5:81-84(1986). The plants may be grown, and either pollinated with the sametransformed strain or different strains, the resulting hybrid havingconstitutive expression of the desired phenotypic characteristicidentified. Two or more generations may be grown to ensure thatexpression of the desired phenotypic characteristic is stably maintainedand inherited, and then seeds harvested to ensure expression of thedesired phenotypic characteristic has been achieved. In this manner, thepresent disclosure provides for transformed seeds (also referred to astransgenic seeds) having a polynucleotide as disclosed herein (e.g., anexpression cassette as disclosed herein) stably incorporated into theirgenome.

Methods for Modulating a Plant Phenotype

In another aspect, the invention relate to a method for modulating aplant phenotype comprising introducing and expressing in a plant apolynucleotide, vector or expression cassette as described above or apolynucleotide encoding a WRKY76 polypeptide as described above. Suchmethods comprise generating from the plant cell a transgenic plant thatexpresses the polynucleotide. In one embodiment, the method comprisesintroducing and expressing a nucleic acid that comprises or consists ofSEQ ID NO:1 or a variant thereof, for example SEQ ID NO:11 or SEQ IDNO:9, a functional variant or part thereof as defined above. In oneembodiment, said polynucleotide has at least 80% sequence identity withthe full-length nucleotide sequence of SEQ ID NO: 1 or a variantthereof, for example SEQ ID NO:11. In another embodiment, saidpolynucleotide has at least 80% sequence identity with the full-lengthnucleotide sequence of SEQ ID NO: 1 or a variant thereof, for exampleSEQ ID NO:11, wherein the polynucleotide contains at least one of:

-   -   (a) adenine as the nucleotide corresponding to position 817 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (b) cytosine as the nucleotide corresponding to position 808 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (c) cytosine as the nucleotide corresponding to position 33 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (d) cytosine as the nucleotide corresponding to position 259 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11; and    -   (e) guanine as the nucleotide corresponding to position 315 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11.

The inventors have surprisingly demonstrated that expressing HaKRKY76increases yield under standard growth conditions and also increasesstress tolerance under mild and severe conditions. Thus, in oneembodiment, said phenotype is increased yield and said method isdirected to increasing yield of a plant compared to a control plant.

The term “yield” includes one or more of the following non-limitativelist of features: early flowering time, biomass (vegetative biomass(root and/or shoot biomass) or seed/grain biomass), seed/grain yield,seed/grain viability and germination efficiency, seed/grain size, starchcontent of grain, early vigour, greenness index, increased growth rate,delayed senescence of green tissue. The term “yield” in general means ameasurable produce of economic value, typically related to a specifiedcrop, to an area, and to a period of time. Individual plant partsdirectly contribute to yield based on their number, size and/or weight.The actual yield is the yield per square meter for a crop and year,which is determined by dividing total production (includes bothharvested and appraised production) by planted square meters.

Thus, according to the invention, yield comprises one or more of and canbe measured by assessing one or more of: increased seed yield per plant,increased seed filling rate, increased number of filled seeds, increasedharvest index, increased viability/germination efficiency, increasednumber or size of seeds/capsules/pods/grain, increased growth orincreased branching, for example inflorescences with more branches,increased biomass or grain fill. Preferably, increased yield comprisesan increased number of grain/seed/capsules/pods, increased biomass,increased growth, increased number of floral organs and/or floralincreased branching. Yield is increased relative to a control plant. Forexample, the yield is increased by 2%, 3%, 4%, 5%-10%, 10%-50% or morecompared to a control plant, for example by at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% or 50%.

In another embodiment, said phenotype is stress tolerance and saidmethod relates to increasing stress tolerance of a plant. The term“stress” or “stress condition” as used herein includes abiotic andbiotic stress. Said stress/stress tolerance is preferably selected fromone or any combination of the following: freezing, low temperature,chilling, drought, high salinity, waterlogging. In one preferredembodiment, the stress is drought.

Species such as winter cereals are adapted to cold or moderate-coldweather and can tolerate temperatures ranging from 0° C. to 15° C., aswell as freezing temperatures, rather well if they have previously beenacclimated to reduced temperatures. By contrast, tropical andsubtropical species, including important crops, such as maize, rice ortomato, are sensitive to low temperatures and appear to lack efficientacclimation mechanisms.

Furthermore, in Arabidopsis research, stress is often assessed undersevere conditions that are lethal to wild type plants. For example,drought tolerance is assessed predominantly under quite severeconditions in which plant survival is scored after a prolonged period ofsoil drying. However, in temperate climates, limited water availabilityrarely causes plant death, but restricts biomass and seed yield.Moderate water stress, that is suboptimal availability of water forgrowth can occur during intermittent intervals of days or weeks betweenirrigation events and may limit leaf growth, light interception,photosynthesis and hence yield potential. Leaf growth inhibition bywater stress is particularly undesirable during early establishment.

In Skirycz et al., 2011, different transgenic Arabidopsis events withenhanced tolerance to lethal drought were analyzed in a mild stressassay. The authors screened the literature in order to identifyArabidopsis genes that in gain- or loss-of-function situations conferdrought stress tolerance without penalties in growth, and then selected25 to perform the assay. In this assay, two lines showed larger plantswhile the rest were smaller, either in control or under droughtconditions. However, growth reduction under mild stress was similar forall of the genotypes tested. The authors therefore concluded thatenhanced survival under severe drought is not a good indicator forimproved growth performance under mild/moderate stress conditions whichcan often be found in temperate climates. Superior survival under severedrought is often associated with constitutive activation of water-savingmechanisms, such as stomatal closure, that can lead to growth penalty.Therefore, genes that are useful in conferring tolerance to severestress conditions in transgenic plants and increase survival rates arein most cases detrimental to plant yield when the transgenic plantexpressing such transgene(s) is exposed to mild stress conditions.

The terms moderate or mild stress/stress conditions are usedinterchangeably and refer to non-severe stress non-lethal stress.Moderate stress, unlike severe stress, does not lead to plant death.Under moderate, that is non-lethal, stress conditions, wild type plantsare able to survive, but show a decrease in growth and seed productionand prolonged moderate stress can also result in developmental arrest.The decrease can be at least 5%-50% or more. The effects of severestress are usually measured as % tage of surviving plants, whereas theeffects of moderate stress can be measured by assessing yield, growth orother parameters other than plant survival. Tolerance to severe stressis measured as a percentage of survival, whereas moderate stress doesnot affect survival, but growth rates.

Accordingly, the term moderate stress as used herein results in ameasurable decrease of growth rates in wild type plants. Assays thatmimic moderate stress conditions for Arabidopsis thaliana plants aredescribed herein and in Skirycz et al, 2011. The decrease may be atleast 5%-50% or more, for example 5%-10%, 1-25%, 20-30%, 30-40%, 40-50%.

The precise conditions that define moderate stress vary from plant toplant and also between climate zones, but ultimately, these moderateconditions do not cause the plant to die. With regard to high salinityfor example, most plants can tolerate and survive about 4 to 8 dS/m.Specifically, in rice, soil salinity beyond ECe˜4 dS/m is consideredmoderate salinity while more than 8 dS/m becomes high. Similarly, pH8.8-9.2 is considered as non-stress while 9.3-9.7 as moderate stress andequal or greater than 9.8 as higher stress.

Drought stress can be measured through leaf water potentials. Generallyspeaking, moderate drought stress is defined by a water potential ofbetween −1 and −2 Mpa. This has for example been applied in experimentsrelating to barley and Phaseolus vulgaris L. (Wingler et al, 1999 andTorres-Franklin et al 2007).

Waterlogging/irrigation: stress. Waterlogging is flooding of the rootsystem whereas submergence is related to immersion of the whole plant(Bailey-Serres et al, Trends in Plant Sciences, 2012, Vo. 17, No. 3,129-138).

Moderate temperatures vary from plant to plant and specially betweenspecies. Normal temperature growth conditions for Arabidopsis aredefined at 22-24° C. For example, at 28° C., Arabidopsis plants grow andsurvive, but show severe penalties because of “high” temperature stressassociated with prolonged exposure to this temperature. However, thesame temperature of 28° C. is optimal for sunflower, a species for which22° C. or 38° C. causes mild, but not lethal stress. In other words, foreach species and genotype, an optimal temperature range can be definedas well as a temperature range that induces mild stress or severe stresswhich leads to lethality.

Also, depending on the soil conditions and/or geographic region in whichthe plant is grown, “mild stress conditions” can be constant/permanent.For example, the yield of the same soybean (or maize) genotype exhibitsdifferences every year when comparing different regions presentingvaried rainfall regimes, even when no drought season occurred duringthis time.

Moderate stress conditions are common even in temperate climates andaffect yield. A skilled person would be able to determine temperaturesthat can lead to mild stress for any given species based on commongeneral knowledge in the technical field and/or routine methods.

Specifically, according of the aspects of the invention, HaWRKY76 can beexpressed in another plant that is not sunflower to elicit thebeneficial effects described herein.

In other aspects, the invention relates to plants or parts thereofobtained or obtainable by the methods described herein as well asproducts derived therefrom.

In yet another aspect, the invention relates to the use of a nucleicacid described above in increasing tolerance to abiotic or bioticstress. In one embodiment, said nucleic acid comprises or consists ofSEQ ID NO:1 or a variant thereof, for example SEQ ID NO: 11 or SEQ IDNO:9, a functional variant or part thereof as defined above. In oneembodiment, said polynucleotide has at least 80% sequence identity withthe full-length nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9. Inanother embodiment, said polynucleotide has at least 80% sequenceidentity with the full-length nucleotide sequence of SEQ ID NO: 1 or avariant thereof, for example SEQ ID NO:11, wherein the polynucleotidecontains at least one of:

-   -   (a) adenine as the nucleotide corresponding to position 817 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (b) cytosine as the nucleotide corresponding to position 808 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (c) cytosine as the nucleotide corresponding to position 33 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11;    -   (d) cytosine as the nucleotide corresponding to position 259 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11; and    -   (e) guanine as the nucleotide corresponding to position 315 of        the full-length nucleotide sequence of SEQ ID NO: 1 or a variant        thereof, for example SEQ ID NO:11.

In one embodiment, said stress is abiotic stress and for exampleselected from drought or irrigation. In one embodiment, said stress ismoderate stress. In another embodiment, said stress is severe stress.

In another aspect, the invention relates to an isolated HaWRKY76promoter nucleic acid sequence. This can be selected from SEQ ID NO: 10or a sequence with at least 80%, 85%, 90%, or 95% sequence identity withSEQ ID NO: 10. In one embodiment, the sequence does not comprise the 5′UTR. In another aspect, the invention relates to a vector comprising SEQID NO: 10 or a sequence with at least 80%, 85%, 90%, or 95% sequenceidentity with SEQ ID NO: 10. Also within the scope of the invention isplant expressing a construct comprising a nucleic acid moleculecomprising SEQ ID NO: 10 or a sequence with at least 80%, 85%, 90%, or95% sequence identity with SEQ ID NO: 10 operably linked to anothernucleic acid sequence, a method for making such plants and uses thereofin controlling stress responses.

Specific embodiments are further described by the following Examples.

While the foregoing disclosure provides a general description of thesubject matter encompassed within the scope of the present invention,including methods, as well as the best mode thereof, of making and usingthis invention, the following examples are provided to further enablethose skilled in the art to practice this invention and to provide acomplete written description thereof. However, those skilled in the artwill appreciate that the specifics of these examples should not be readas limiting on the invention, the scope of which should be apprehendedfrom the claims and equivalents thereof appended to this disclosure.Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.All documents mentioned in this specification are incorporated herein byreference in their entirety.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein. Unless context dictates otherwise, the descriptionsand definitions of the features set out above are not limited to anyparticular aspect or embodiment of the invention and apply equally toall aspects and embodiments which are described. The application claimsbenefit from U.S. 61/986,730. HaWRKY76 is termed HaWKKY2 therein.Accordingly, the terms HaWRKY76 and HaWKKY2 are synonymous and are usedinterchangeably.

EXAMPLES

In reference to the following Examples, it will be recognized by personsskilled in the art that a transcription factor that is associated with aparticular first trait may also be associated with at least one other,unrelated and inherent second trait which is not predicted by the firsttrait.

Genetic Construct

35SCaMV:HaWRKY76 was employed in the various Examples described below.The HaWRKY76 coding sequence was obtained from Helianthus annuus CF31genotype. It was amplified by RT-PCR with specific oligonucleotidesusing as a template the total RNA of the leaves. The amplificationproduct was cloned directly in the pBI121 vector. In this way, the cDNAexpression was controlled by the 35S CaMV promoter. The construct35S:HaWRKY76 was initially introduced in the BL21 (DE3) E. coli strainand then transferred to Agrobacterium tumefaciens strain LBA4404 byelectroporation using the GENE PULSER™ (Bio-Rad). The protein sequenceas shown in SEQ ID NO:12 was expressed in plants.

Plant Material and Growth Conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was purchased from LehleSeeds (Tucson, Ariz.). Plants were grown directly on soil in a growthchamber at 22-24° C. under long-day photoperiods (16 hours ofillumination with a mixture of cool-white and GroLux fluorescent lamps)at an intensity of approximately 180 μE m⁻²s⁻¹ in pots (8 cm diameter×7cm height).

Seeds of Helianthus annuus L. (sunflower CF31, from Advanta Seeds) weregrown in the soil pots in a culture chamber at 28° C. for variableperiods of time depending on the purpose of the experiment, as furtherdetailed in the figure legends.

Drought Assays

Depending on the particular Experiment, as detailed in the figurelegends, sunflower seedlings were placed on a filter paper during 30minutes, and R2 plants grown in soil pots were stressed by stoppingwatering (dehydration) during 15 days. Every one, two, or three daysafter this treatment, 1 cm diameter leaf disks were frozen in liquidnitrogen for further RNA analysis.

As a control of viability of the plants, plants were re-watered afterthe taking of the sample. Only the samples of the survivors wereanalyzed.

25-day-old Arabidopsis plants were subjected to drought stress bystopping of watering (dehydration) during 16-18 days. After thistreatment, the plants were re-watered to saturation. Survivor plantswere counted two days after recovery at normal growth conditions.

In the case of drought assays maintaining the same water volume, soilpots were weighed every 2-3 days, and water was added to those pots thatneeded watering in order to maintain the same weight in each soil pot.The weight differences were recorded so as to calculate the total watervolume added to each pot.

Transformation of Arabidopsis thaliana

Transformed Agrobacterium tumefaciens strain LBA4404 was used to obtaintransgenic Arabidopsis plants. The method employed for transformingArabidopsis thaliana was the one using immersion (the floral dipprocedure), as describe by Clough and Bent, Plant J. 16:735-743 (1998).Transformed plants were selected on the basis of kanamycin resistanceand positive PCR, which was carried out on genomic DNA with specificoligonucleotides. Five positive independent lines were used to selecthomozygous T3 and T4 in order to analyze phenotypes and the expressionlevels of HaWRKY76. To assess HaWRKY76 expression, real time RT-PCR wasperformed on homozygous transformants, as described further below.Plants transformed with pBI101.3 were used as negative controls.

RNA Isolation and Expression Analyses by Real Time RT-PCR

RNA for real time RT-PCR was prepared with TRIZOL® reagent (Invitrogen™)according to the manufacturer's instructions. RNA (2 μg) was used forthe RT-PCR reactions using M-MLV reverse transcriptase (Promega).Quantitative PCRs were carried out using a MJ-Chromo4 apparatus in a 20μl final volume containing 1 μl SyBr green (10×), 9 pmol of each primer,2 mM MgCl₂, 10 μl of a 1/30 dilution of the RT-PCR reaction, and 0.05 μkPlatinum Taq (Invitrogen Inc.). Fluorescence was measured at 78-80° C.during 40 cycles. Sunflower total RNA was also prepared with the TRIZOL®(Invitrogen Inc.) technique.

Chlorophyll Quantification

Extracts from 100 weighed mg of leaves were prepared after freezing withliquid nitrogen. 1.5 ml of 80% acetone was added to each sample, and thetubes were placed in darkness for a period of 30 minutes. During thisincubation, the sample solids were decanted and the absorbance at 645and 663 nm, respectively, was measured in the supernatants with aspectrophotometer. Chlorophyll concentrations were quantified accordingto the methods disclosed in Whatley et al. (1963).

Injury Evaluation by the Ion Leakage Technique

The ion leakage technique was carried out essentially as described bySukumaran and Weiser (1972) with minor modifications. The conductivitypercentage was calculated as the ratio between C2/C1, i.e.,[L=(C1/C2)*100], and was used as the index of injury. C1 represents theconductance after the treatment, and C2 represents the maximumconductance of each sample. L values higher than 50% indicate a severeinjury.

Water Loss Evaluation

Fully expanded leaves were detached from Wild-Type (WT) and the35S:HaWRKY76 transgenic plants, respectively, and placed on Petri dishesunder strictly controlled conditions. Water loss was determined byweighing the leaves at the times indicated in the Figures as comparedwith the initial weight. The values are reported as a percentage of theinitial weight.

Example 1

Seeds of Helianthus annuus L. (sunflower CF31, from Advanta Seeds) weregrown in the soil pots in a culture chamber at 28° C. for a period of 5days. The HaWRKY76 transcription levels were quantified by RT-PCR andnormalized with housekeeping ACTIN transcripts. The obtained values werenormalized with respect to the value measured in the root sample, whichwas arbitrarily assigned a value of 1. An Anova test was performed,followed by a Fisher LSD post-hoc test. The results are shown in FIG. 5.The different letters (a and b) indicate samples that were found to besignificantly different (i.e., p value<0.05). As demonstrated by theresults summarized in FIG. 5, HaWRKY76 expression patterns in 5-day-oldsunflower seedlings indicated higher levels in roots and hypocotyls thanin cotyledons.

Example 2

Seeds of Helianthus annuus L. (sunflower CF31, from Advanta Seeds) weregrown in the soil pots for 5 days under control conditions and thenplaced for 30 minutes on a filter paper (Drought) or in MS 0, 5×solution (Control). The HaWRKY76 transcription levels were quantified byRT-PCR and normalized with housekeeping ACTIN transcripts. The obtainedvalues were normalized with respect to the value measured in the controlroot sample, which was arbitrarily assigned a value of 1. An Anova testwas performed, followed by a Fisher LSD post-hoc test. The results areshown in FIG. 6A. The different letters (a, b, and c) indicate samplesthat were found to be significantly different (i.e., p value<0.05).

Sunflower R2 plants were subjected to a continuous and severe droughtstress (stopping watering during 15 days). The leaf disks were frozen atthe period of time indicated in FIG. 6B. As a control of the plants'viability, the plants were re-watered after sampling. The transcriptionlevels of HaWRKY76 were quantified in these sunflower R2 plantssubjected to a continuous and severe drought stress. The obtained valueswere normalized with respect to the value measured in the sampleharvested at day 2 which was arbitrarily assigned a value of 1. An Anovatest was performed, followed by a Fisher LSD post-hoc test. The resultsare shown in FIG. 6B. As demonstrated by the results summarized in FIG.6, HaWRKY76 expression is up-regulated by water stress.

Example 3

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.Transcription levels of HaWRKY76 were quantified by real time RT-PCR.All of the values were normalized with respect to the value measured inline W76-C, which was arbitrarily assigned a value of 1 and consideredas a low-level-expression line. WT plants were used as negativecontrols, and error bars correspond to the standard deviations fromthree biological replicas. An Anova test was performed, followed by aFisher LSD post-hoc test. FIG. 7 shows the relative expression levels ofHaWRKY76 in Arabidopsis transgenic plants bearing the construct35S:HaWRKY76. Different letters (a, b, and c) indicate samples that werefound to be significantly different (i.e., p value<0.05).

Example 4

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The plants were grown in standard growth conditions. The root length wasmeasured in the 35S:HaWRKY76 (W76-A, W76-B and W76-C) and the WT plants.An Anova test was performed, followed by a Fisher LSD post-hoc test.Error bars correspond to the standard deviations twenty biologicalreplicas.

The average root lengths and standard deviations were calculated in 7-and 14-day-old seedlings. The averages of each genotype were calculated,and the results are shown in panel (A) of FIG. 8. Different letters (a,b, c, and d) indicate samples that were found to be significantlydifferent (i.e., p value<0.05). Panel (B) of FIG. 8 is a photograph ofthe roots of the 7-day-old plants grown on Petri dishes. As demonstratedby the results shown in FIG. 8, Arabidopsis plants bearing the construct35S:HaWRKY76 develop longer roots than the control plants in standardgrowth conditions.

Example 5

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.Roots were detached from 33-day-old 35S:HaWRKY76 (W76-A, W76-B andW76-C) and WT control plants grown on sand in standard conditions, andthe average weights of the respective roots were measured. An Anova testwas performed, followed by a Fisher LSD post-hoc test. Error barscorrespond to the standard deviations from three biological replicas.

The average root weight of each genotype is shown in FIG. 9. Differentletters (a and b) indicate samples which were found to be significantlydifferent (i.e., p value<0.05). As demonstrated by the results shown inFIG. 9, Arabidopsis plants bearing the construct 35S:HaWRKY76 grown onsand in standard growth conditions exhibit higher root biomass comparedwith WT control plants.

Example 6

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The plants were grown on poor soil in standard growing conditions.Rosettes of 36-day-old 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WTplants were detached, and the weight of the rosettes was measured. AnAnova test was performed, followed by a Fisher LSD post-hoc test. Errorbars correspond to the standard deviations from four or five biologicalreplicas. The rosette weight (mg) of each genotype is shown in panel (A)of FIG. 10.

The weighed leaves of the 36-day-old plants were frozen, and chlorophyllwas extracted with acetone and quantified by absorbance at 645 and 663nm, respectively (see “Chlorophyll Quantification” conditions above).The total chlorophyll content per plant is shown in panel (B) of FIG.10. The protein content was also determined using BSA as standardaccording to the method described in Bradford (1976). The proteinconcentration per plant is shown in panel (C) of FIG. 10.

As demonstrated by the results shown in FIG. 10, Arabidopsis plantsbearing the construct 35S:HaWRKY76 grown on poor soil in standardconditions exhibit higher rosette biomass compared with the WT controlplants. Different letters (a and b) indicate samples which were found tobe significantly different (i.e., p value<0.05). As also shown in FIG.10B and FIG. 10C, the chlorophyll content (μg) per mg leaves is similarwhen comparing genotypes. However, because rosettes of the transgenicplants are larger, the total protein and chlorophyll contents differwhen comparing genotypes, i.e., the contents are larger in thetransgenic plants than in the WT control plants.

As further shown in FIG. 11, 35-day-old Arabidopsis plants bearing theconstruct 35S:HaWRKY76 grown in standard conditions exhibit higheraerial biomass compared with the WT control plants. Error barscorrespond to the standard deviations from four biological replicas.

Example 7

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The plants were grown on poor soil in standard growing conditions. Theseeds of each plant from each of the 35S:HaWRKY76 (W76-A, W76-B andW76-C) and WT genotype were separately harvested and weighed. Theaverage yield of 4 plants of each genotype is shown in FIG. 12A. AnAnova test was performed, followed by a Fisher LSD post-hoc test.Different letters (a and b) indicate samples which were found to besignificantly different (i.e., p value<0.05).

FIG. 12B shows the yield per plant, with the horizontal linerepresenting the average value of the WT plants. FIG. 12C is aphotograph of the harvested seeds of 4 plants of each genotype. Asdemonstrated by the results shown in FIG. 12, Arabidopsis plants bearingthe construct 35S:HaWRKY76 grown in poor soil conditions exhibit higheryields than the WT control plants.

As additionally shown in FIG. 13, Arabidopsis plants bearing theconstruct 35S:HaWRKY76 grown on standard conditions also exhibit higheryields than the WT control plants. Specifically, FIG. 13 shows theaverage yield of 4 plants of each genotype. An Anova test was performed,followed by a Fisher LSD post-hoc test. Different letters (a and b)indicate samples which were found to be significantly different (i.e., pvalue<0.05).

Example 8

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were wellirrigated for 25 days. Then, the 25-day-old well-irrigated plants weresubjected to drought stress by stopping watering until severe damage wasvisible. At that time, the plants were re-watered. FIG. 14 is aphotograph of the plants taken 4 days after the re-watering.

As can be seen in the photograph of FIG. 14, Arabidopsis plants bearingthe construct 35S:HaWRKY76 are more tolerant to drought than their WTcontrol plants.

Example 9

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown onpoor soil (undefined stress), and were subjected to drought during thevegetative stage and also during the reproductive stage. The plants weresubjected to drought stress by stopping watering until severe damage wasvisible. At that time, the plants were re-watered.

FIG. 15A is a photograph of the plants subjected to drought during thevegetative stage, and FIG. 15C is a photograph of the plants subjectedto drought during the reproductive stage.

The yield (seed production) of the plants was measured. Error barscorrespond to the standard deviations from four biological replicas. AnAnova test was performed, followed by a Fisher LSC post-hoc test. FIG.15B shows the yield of the plants when subjected to drought during thevegetative stage, and FIG. 15D shows the yield of the plants whensubjected to drought during the reproductive stage. Different letters (aand b) indicate samples that were found to be significantly different(i.e., p value<0.05).

As demonstrated by the results shown in FIG. 15, Arabidopsis plantsbearing the construct 35S:HaWRKY76 grown in poor soil conditions aremore tolerant to drought than their WT control plants and show enhancedyield when the stress is applied during the vegetative stage. When thestress was suffered during the reproductive stage, no significantdifferences between the genotypes were observed.

The top panel of FIG. 16 shows the enhanced yield of the transgenicplants when the drought stress was suffered during the vegetative stage,while the lower panel shows that no significant differences in yieldbetween the genotypes were observed when the drought stress was sufferedduring the reproductive stage. In FIG. 16, error bars correspond to thestandard deviations from four biological replicas.

Similar experiments were conducted in a second set of experiments andthe results are shown in FIG. 35.

Example 10

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W6-B and W76-C) and WT plants were grown onsoil and normal watering stopped when the plants were 25-days old (milddrought stress). Soil pots (poor soil) were weighed every two daysduring the assay and water was added to those that needed it to maintainthe same weight in all pots. After harvesting, plant yield was evaluatedfor each individual plant of each genotype.

FIG. 17A shows the average amount (ml) of water added to the soil foreach genotype, and FIG. 17B shows the average yield per plant of eachgenotype. An Anova test was performed, followed by a Fisher LSC post-hoctest. Error bars correspond to the standard deviations from 10biological replicas. Different letters (a and b) indicate samples thatwere found to be significantly different (i.e., p value<0.05).

As demonstrated by the results shown in FIG. 17, Arabidopsis plantsbearing the construct 35S:HaWRKY76 need less water than the WT controlplants during a mild drought stress exhibiting similar yields.

Example 11

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were wellirrigated (FIG. 18A) or subjected to moderate drought stress (FIG. 18B).In both cases, fully expanded leaves were detached from the 35S:HaWRKY76and the WT plants that were (A) well irrigated and (B) subjected tomoderate drought stress. The detached leaves were placed on Petri dishesunder controlled conditions and weighed at the times indicated in FIGS.18A and 18B, respectively. The values are reported as weight at eachmeasured time with respect to the initial weight.

FIG. 18A shows the weight of leaves (% of initial weight) of thewell-irrigated plants, FIG. 18B shows the weight of leaves (% of initialweight) of the plants subjected to moderate drought stress, and FIG. 18Cis a photograph taken after 8 hours of treatment. An Anova test wasperformed, followed by a Fisher LSC post-hoc test. Error bars correspondto the standard deviations from 3 or 4 biological replicas. Differentletters (a and b) indicate samples that were found to be significantlydifferent (i.e., p value<0.05).

As demonstrated by the results shown in FIG. 18, Arabidopsis plantsbearing the construct 35S:HaWRKY76 lost less water than their WT controlplants.

Example 12

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown onpoor soil for 25 days. The 25-day-old plants were then completelysubmerged during 5-6 days and placed in standard growing conditionsuntil recovery. The number of survivors after treatment and the averageseed yield of the survivors were calculated. One 6-day submergenceexperiment was performed with 12 plants of each genotype. As the numberof survivors was not equal for all genotypes, the seed yield ofsurvivors was calculated from one 5-day submerge experiment performedwith 8 plants of each genotype.

FIG. 19A shows the percentage of survivors of each genotype after thetreatment. FIG. 19B is a photograph of the plants taken after one weekof recovery. FIG. 19C shows the average seed yield of survivors. AnAnova test was performed, followed by a Fisher LSC post-hoc test. Errorbars correspond to the standard deviations from 4-9 biological replicas.Different letters (a and b) indicate samples which were found to besignificantly different (i.e., p value<0.05). FIG. 19D is an image ofseeds produced by each genotype with respect to the average seed yieldsshown in FIG. 19C.

As demonstrated by the results shown in FIG. 19, Arabidopsis plantsbearing the construct 35S:HaWRKY76 are more tolerant to submergence andexhibited higher yields after such submergence than their WT controlplants.

The 25-day-old 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plantssubjected to submergence during 5-6 days as described above were furtherevaluated according to different parameters after recovery.Specifically, the rosette weight of the respective plants was measured 2and 5 days after the start of treatment (submergence), and the resultsare shown in FIG. 20A. FIG. 20B is a photograph of the rosettes taken 5days after the start of treatment. The chlorophyll content of the plantswas also measured at 2 and 5 days after the start of treatment, and theresults are shown in FIG. 20C. Finally, the total content of solublesugars (μg/mg leaves) was determined for each of the plants, and theresults are shown in FIG. 20D. With respect to the results shown inFIGS. 20A, 20C, and 20D, an Anova test was performed, followed by aFisher LSD post-hoc test. Error bars correspond to the standarddeviations from four biological replicas. Different letters indicatesamples that were found to be significantly different (i.e., pvalue<0.05).

Example 13

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.Soluble carbohydrates and starch were enzymatically determined in therosettes of 25-day-old 35S:HaWRKY76 and WT plants that had beensubmerged during 2, 3, or 5 days.

FIG. 21A shows the soluble glucose per mg of fresh rosette weight in foreach of the plants. FIG. 21B shows the sucrose content in the respectiveplants, and FIG. 21C shows the starch content in the respective plants.As can be seen from the results of FIG. 21, the transgenic plants havemore soluble carbohydrates during submergence than the WT controlplants. Error bars correspond to the standard deviations from 4biological replicas. An Anova test was performed, followed by a FisherLSC post-hoc test. Different letters (a, b, c, and d) indicate samplesthat were found to be significantly different (i.e., p value<0.05).

Example 14

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.25-day-old transgenic plants bearing the construct 35S:HaWRKY76 and WTplants were completely submerged during 5 days.

FIG. 22A shows the protein content per mg of fresh rosette weight of therespective plants, and FIG. 22B shows the chlorophyll content per mg offresh rosette weight of the respective plants. As can be seen from thequantified results in FIG. 22, the transgenic plants exhibit higherprotein concentrations than the WT control plants during submergence.Error bars correspond to the standard deviations from 4 biologicalreplicas. An Anova test was performed, followed by a Fisher LSC post-hoctest. Different letters (a, b, c, and d) indicate samples that werefound to be significantly different (i.e., p value<0.05).

Example 15

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 transgenic plants and WT plants were grown on sand for35 days. On day 35, the complete root system was collected and weighed.

FIG. 23A shows the root/aerial biomass ratio for each of the respectiveplants, and FIG. 23B shows the protein content of the roots collectedfrom each of the respective plants. As can be seen from the results inFIG. 23, the transgenic plants show a higher root/aerial tissue biomassratio than the WT control plants. Error bars correspond to the standarddeviations from 7 biological replicas. An Anova test was performed,followed by a Fisher LSC post-hoc test. Different letters (a, b)indicate samples that were found to be significantly different (i.e., pvalue<0.05).

Example 16

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.25-day-old 35S:HaWRKY76 transgenic plants and WT plants were subjectedto waterlogging during 5 days. Transverse sections of stems of therespective plants were then obtained and stained. The results are shownin the photographs of FIG. 24. As can be seen from FIG. 24, thetransgenic plants exhibit more aerenchyma than the WT control plants.

Example 17

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were initiallygrown in standard growing conditions, then subjected to submergenceduring 5 days, and finally recovered during one additional day. Thesucrose content of the respective plants was measured.

FIG. 25 shows the sucrose content expressed as μg of glucose/mg ofleaves, with each bar representing the average value obtained from 4plants. An Anova test was performed, followed by a Fisher LSC post-hoctest. Error bars correspond to the standard deviations from 4 biologicalreplicas. Different letters (a, b, c, and d) indicate samples that werefound to be significantly different (i.e., p value<0.05).

As demonstrated by the results shown in FIG. 25, the transgenic plantsexhibit higher sucrose content after submerge than the WT controlplants.

Example 18

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were initiallygrown in standard growing conditions. Thereafter, 25-day-old plants ofeach genotype were completely submerged during 5 days and placed instandard growing conditions until recovery. The average yield wasmeasured in 5 plants per genotype.

The results are shown in FIG. 26. An Anova test was performed, followedby a Fisher LSC post-hoc test. Error bars correspond to the standarddeviations. Different letters (a and b) indicate samples that were foundto be significantly different (i.e., p value<0.05).

As demonstrated by the results shown in FIG. 26, the transgenic plantsexhibited slightly higher yields than the WT control plants after 5 daysof submergence.

Example 19

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown onsoil for 25 days. Then, the 25-day-old plants of each genotype weresubjected to waterlogging treatment during one week. The tolerance ofthe respective plants to the waterlogging treatment was measured withrespect to the rosette weight, membrane stability, and stem length.

Panel (A) of FIG. 27 shows the rosette weights of the plants measured 6and 7 days after starting the waterlogging treatment. Panel (B) of FIG.27 shows the membrane stability (% of ion leakage) measured 5 and 7 daysafter starting the treatment. Panel (C) of FIG. 27 shows the stemlengths of the plants after the waterlogging treatment, measured with aruler. An Anova test was performed, followed by a Fisher LSD post-hoctest. Error bars correspond to standard deviations from 4 biologicalreplicas. Different letters indicate samples that were found to besignificantly different (i.e., p value<0.05).

As demonstrated by the results shown in FIG. 27, the 35S:HaWRKY76transgenic plants are more tolerant to waterlogging than the WT controlplants.

Example 20

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown onpoor soil for 25 days. Then, the 25-day-old plants of each genotype weresubjected to waterlogging treatment during one week. The 35S:HaWRKY76and WT plants' seeds were harvested and weighed after the waterloggingtreatment. All of the plants survived the waterlogging treatment.

Panel (A) of FIG. 28 shows the average yield per genotype. Panel (B) ofFIG. 28 shows the yield of each plant, the horizontal line representingthe average value of the WT plants. An Anova test was performed,followed by a Fisher LSD post-hoc test. Error bars correspond tostandard deviations from 4 biological replicas. Different letters (a andb) indicate samples that were found to be significantly different (i.e.,p value<0.05).

As demonstrated by the results shown in FIG. 28, the 35S:HaWRKY76transgenic plants exhibit higher yields after waterlogging than the WTcontrol plants.

Example 21

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown instandard soil, and then subjected to a waterlogging treatment during oneweek. All of the plants survived the waterlogging treatment.

Following the waterlogging treatment, seeds of the 35S:HaWRKY76 and WTplants were harvested and weighed. The top panel of FIG. 29 shows theaverage yield per genotype, whereas the bottom panel of FIG. 29 showsthe yield of all the plants of each genotype. An Anova test wasperformed, followed by a Fisher LSD post-hoc test. Error bars correspondto standard deviations from 4 biological replicas. Different letters (aand b) indicate samples that were found to be significantly different(i.e., p value<0.05).

As demonstrated by the results shown in FIG. 29, the 35S:HaWRKY76transgenic plants exhibit higher yields after a week of waterloggingthan the WT control plants.

Example 22

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown instandard growing conditions.

The number of rosette leaves of the 35S:HaWRKY76 and WT plants wascounted, and the results are shown in FIG. 30. Error bars correspond tothe standard deviations from eight biological replicas. As demonstratedby the results shown in FIG. 30, when grown in standard conditions, thetransgenic plants do not exhibit significant differences in the numberof leaves/rosettes in comparison to the WT control plants.

Example 23

Transgenic Arabidopsis thaliana plants bearing the construct35S:HaWRKY76 were obtained by the floral dip method (see “Constructs”above), and three homozygous lines were selected for further analysis.The 35S:HaWRKY76 (W76-A, W76-B and W76-C) and WT plants were grown instandard growing conditions.

The stem lengths of the 35S:HaWRKY76 and WT control plants was measuredduring their life cycle. Specifically, the stem lengths were measuredevery 3-5 days starting during the plants' transition from thevegetative to the reproductive stage. The results are shown in FIG. 31.Error bars correspond to the standard deviations from eight biologicalreplicas. As demonstrated by the results shown in FIG. 31, when grown instandard conditions, the 35S:HaWRKY76 transgenic plants do not exhibit adelay in their life cycle in comparison to the WT control plants.

Example 24

FIG. 32 is an image of an EMSA assay performed with purified recombinantHaWRKY76-GST. Lines 1 and 2 are 20 ng of W2-GST with a 12 N labelednucleotide (10000 and 17000 cpm respectively). Line 4 is 20 ng of W2-GSTwith 10000 cpm of labeled oligonucleotide exhibiting 2 W-Boxes(C/TTGACT/C) separated by 5 nucleotides. Lines 5 and 6 are negativecontrols without the protein and with the same amount of labeledoligonucleotides used in lines 2 and 4 respectively.

The results of the assay show that the recombinant protein HaWRKY76binds with more affinity an oligonucleotide with a central core 12 N (4nucleotides per position) than a selected oligonucleotide exhibiting twoW-Boxes separated by 5 nucleotides. This indicates a different bindingaffinity than their related WRKY proteins.

INCORPORATION BY REFERENCE

All references and nucleotide and polypeptide sequences cited are herebyincorporated by reference in their entireties herein.

SEQUENCE INFORMATION HaWRK76 cDNA SEQ ID NO: 1atggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgtttttcaaacaaaccggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagccaaagtgaa HaWRK76 protein SEQ ID NO: 2MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHARFRRGPSSTTGDTNGPSTSSHSEGKSRDTTSFVQNECFSNKPVTEITTTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSSLGIVAPAPTFSSRKPPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVPITGSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARKRVELSADDSKMLIVTYDGEHRHRDRHAPVPMSLIGVYGEPK HaT131007971 GENE SEQ ID NO: 3ccccatctacccctcatatctctcatcaatctctctttctctctcttagggtttcaacttccaaccttcttctacaacaatggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcttcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagtcaaagtgagggggacacatgtgtggtccgtgagcactttgcacagttttctaaggtcaacaggaagagagagaaaataactttttttattcttggtttagttgagggttaatttgtacatttgacaaaagatgaagggtgtaattggtaatttagaagatgcccccagatctgatattcgattttgtttggactaattactttataaaagttgatattggtatatttaaaatttaattaaagaggaaaagtaattagtccaaacaaaatcgaatatcagatctgHaT131007971 PROTEIN SEQ ID NO: 4Met Ala Val Asp Phe Val Gly Ile Gin Ser Thr Asp His Leu Leu Asn Arg Met Phe Gln LeuLeu Ser His Asp Leu Asn Val Ser Ser Thr Tyr Thr His Ala Val Ser Ala Phe Lys Arg Thr GlyHi's Ala Arg Phe Arg Arg Gly Pro Ser Ser Thr Thr Gly Asp Thr Asn Gly Pro Ser Thr Ser SerHis Ser Glu Gly Lys Ser Arg Asp Thr Thr Ser Phe Val Gin Asn Glu Cys Phe Ser Asn Lys SerVal Thr Glu Ile Thr Thr Thr Thr Thr Ser Thr Ser Ser Ser Ser Val Val Ser Ser Ser Thr Gly GlyAsn Leu Asp Gly Ser Val Ser Asn Gly Lys Gln Phe Ser Ser Leu Gly Ile Val Ala Pro Ala ProThr Phe Ser Ser Arg Lys Pro Pro Leu Pro Ser Thr His Arg Lys Arg Cys Gly Ala Asp Arg ProVal Ala Ser Val His Gly Ser Gly Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly SerLys Arg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser Ile Pro Ala Asp Asp Tyr SerTrp Lys Lys Tyr Gly Glu Lys Lys Ile Asp Gly Ser Leu Tyr Pro Arg Val Tyr Tyr Lys Cys IleThr Gly Lys Gly Cys Pro Ala Arg Lys Arg Val Glu Leu Ser Ala Asp Asp Ser Lys Met Leu IleVal Thr Tyr Asp Gly Glu His Arg His Arg Asp Arg His Ala Pro Val Pro Met Ser Leu Thr GlyVal Tyr Gly Glu Ser Lys HuCL13748C001 GENE SEQ ID NO: 5cttggcggggatcatctctctttctctctctctctcttagggtttcaacttccaaccttcttctacaacaatggcggttgatttcgtcggaattcaatctacagatcatcatctaaaccgcatgtttcagttatcaactcacgatttaaacgtttcgtcaacctacacacacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgagatacgacgtcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagtttttttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgctaccgtcgactcatcggaaaaggtgcagcgctgatcgtcctgttgcttccgtacacggatctggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtgtattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgtgccggtacttatgagtttgaccggtgtgtatggtgagtcaaagtgagggggacacatgtgt HuCL13748C001 PROTEIN SEQ ID NO: 6Met Ala Val Asp Phe Val Gly Ile Gin Ser Thr Asp His His Leu Asn Arg Met Phe Gin Leu SerThr His Asp Leu Asn Val Ser Ser Thr Tyr Thr His Ala Val Ser Ala Phe Lys Arg Thr Gly HisAla Arg Phe Arg Arg Gly Pro Ser Ser Thr Thr Gly Asp Thr Asn Gly Pro Ser Thr Ser Ser HisSer Glu Gly Lys Ser Arg Asp Thr Thr Ser Phe Val Gln Asn Glu Cys Phe Ser Asn Lys Ser ValThr Glu Ile Thr Thr Thr Thr Thr Ser Thr Ser Ser Ser Ser Val Val Ser Ser Ser Thr Gly Gly AsnLeu Asp Gly Ser Val Ser Asn Gly Lys Gin Phe Phe Ser Leu Gly Ile Val Ala Pro Ala Pro ThrPhe Ser Ser Arg Lys Pro Pro Leu Pro Ser Thr His Arg Lys Arg Cys Ser Ala Asp Arg Pro ValAla Ser Val His Gly Ser Gly Ser Gly Cys His Cys Cys Ser Lys Arg Arg Lys Thr Gly Ser LysArg Glu Ile Arg Arg Val Pro Ile Thr Gly Ser Lys Ile Thr Ser Ile Pro Ala Asp Asp Tyr Ser TrpLys Lys Tyr Gly Glu Lys Lys Ile Asp Gly Ser Leu Tyr Pro Arg Val Tyr Tyr Lys Cys Ile ThrGly Lys Gly Cys Pro Ala Arg Lys Arg Val Glu Leu Ser Ala Asp Asp Ser Lys Met Leu Ile ValThr Tyr Asp Gly Glu His Arg His Arg Asp Arg His Val Pro Val Leu Met Ser Leu Thr Gly ValTyr Gly Glu Ser Lys SEQ ID NO: 7 WKKYGEK SEQ ID NO: 8 WRKYGQKHaWRK76 genomic DNA SEQ ID NO: 9aatacaattgattactcagtcgaataatggccaatatccgttcataatcgacgacgacgtcactagggttttttctcttccggttaccaggcctatcatctggtttctggtttattttgagggtgagggttttgcattgattcctacgctccaatcatctgcttcctgaattgaatctgaatctgaatctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagattccaagcattttatcgtgtgttgtaattttaatgaagcaatgaagattgaatatgcactagggtgagggttttgcattgattcctgcgctccaatcatctgcttcctgaattgaatctgaatctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagaagagtatgttgatataggagagttgaacccgacacgtcttctgtgatgattcaaaacattccgaataactagcagaacccgatccacttacattcgaataacgctgagaatccacaacgcttatggccagattctttccttcaatcttggcaaaatcaatgcctgcttgttttattatcacgaaagtactgaacaaccgagataggcacaataacaacaacagctttccatgatttggagactgatcggaatcgaatcagtgattttactggaagccgtttgattatgttttattatcacgaaagtactgaacaaccgagatagacacaataacaacaacaactttccatgatttggagactgatcagaatcgaaacagtgattttactttactggaagccgtttgattatttcctcttggatctcgaaaggcacgttgtctgacatcttgattttgattaccaactcaccatatcaatttctaggtagttaacaacgctaattagaaaaaaaaacgtgattcatatacacaacggtctctttgcttcgtccactagaactatttgtgtatgcattcttttggtagttaacaacgctaattagaaaaaaatctgtaaaattaatcatatcgattgctataacaggatggatgtgacacacacagatagataagctgtcacagttgtcaagactgtttgtcgggtttccaatgctcacacagggtgataaacaatgctgatacgaacacatgtgtttcgaaataggctttgttcttggtttaatgaagttacatgtaacatctacttatattcagatttaaacaatgcccgcctcaactgagataacggacatccatcaagctcaaaaagaaaacttgtgtttcccatagtcacaaaagattgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgggtttgcacattcaatcttcaatacttcattaaaataccactagtagaagaacccgacacgtcttcatgttacggagaatgttctacaaattgaaggaaaaggaaaagatttgttgaaatcgaatttgacaactatgacagcttattaaaagtagcaatcacctccaatcatttcctggtcaaatcacacaccataaagtttaaagttatcgtgtgttgtagtttggaaagatgaccgaacaaagatctgaataacaaactgtggagaacttgtttgtgtcgggttcttctgctagttacttgatcaacggactgatcgttaacgttactctacttgtgctccgatttaagcggcgtgttccgatcaacgttgtttaaaggtgatgaagagtttagaaaccctagaatgatggtgcttttacgggaaaaaacgtgataattgaaaaacatcaatctatacggcctgtatacaattatacggcccgtatacatttggtaaacatcaaaaacctcccaaaataaatccattgagccgtatagtttttacaaatcgtatacacatgtatacggctcgtataactatatacgggccgtatatagtaaaaaaaacattctttacgcattgtcctattaaaaaacattctatacggaccgtatattttgtatacggtccgtataaactaaagatatgaaaataaaatatagggggtgtgggaataagaggagcgttattaaaatacacaaataattttcgtcatgactccaactacatgttttattgtgttcttgtttgtcgttattaatattttctttgacatagaaagtcgaaacgcatggtatccagctacgtacacatttattcaacttatatttattttagtcctcgtttgactttatgcctccactagctttctccatctgtttatcacgctgcaaataagtcaaacaattctccaccgtccgatcaaaacagtatctcattgactccactttcccaattggacctttataacccccttctacccctcatatctctcatcaatctctctttctctctcttagggtttcaacttccaaccttcttctacaacaatggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgagatacgacttcgtttgtacaaaacgagtgtttttcaaacaaatcggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcttcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgccattgggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagtcaaagtgagggggacacatgtgtggtccgtgagcactttgcacagttttctaaggtcaacaggaagagagagaaattaactttttttattcttggtttagttgagggttaatttgtacatttgacaaaagatgaagggtgtaattggtaatttagaagatgccccagatctgatattcgattttgtttggactaattactttataaaagttgatattggtatatttaaaatttaattaaagaggaaaagtaatttgaataagtttgatgccacagcagagtcaatgggttttaaagtctctttaaatgactaaaaaattagataattgaatgaattttttaatggcaaatgtagtctttattttcatatttattatggtcgttggtgcgacttttggcaaattgatttcgacaatgtattgatggcgatgatctggaatggtccaatccaattttttatttgttttattgtttaatatttaggagactttggaaaaatagcaagggttgaccctgatgaatattaataagttgtgtttactaaagaagcaagaatgtaacagctagcgatgagatgttaactaacgggtaccgtattgatgtcgagctaaaaccaaaaccaaataacataatgtgtatgttcagttggtattggtattatacggtaccgctaaaaatcccaaacaggtaagtgccacatggtatcgatactgcagcttagataaaataaaattgaatatcacaccgtatatgtttggtcgacatcaataacaggtattcggtatgaagtttcccatctcaagactggcacgtaatatcatatatatacaactatagttggttttatactttcggagtttacggtttcaacttctattagttgggtgaagagcatccaagaggtcatcaatc tgtaHaWRK76 promoter 5′UTRs underlined SEQ ID NO: 10aatacaattgattactcagtcgaataatggccaatatccgttcataatcgacgacgacgtcactagggttttttctcttccggttaccaggcctatcatctggtttctggtttattttgagggtgagggttttgcattgattcctacgctccaatcatctgcttcctgaattgaatctgaatctgaatctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagattccaagcattttatcgtgtgttgtaattttaatgaagcaatgaagattgaatatgcactagggtgagggttttgcattgattcctgcgctccaatcatctgcttcctgaattgaatctgaatctgaaagttaactagacccctcaagttgtcattcgtaacaactaagcgtctggagaagagtatgttgatataggagagttgaacccgacacgtcttctgtgatgattcaaaacattccgaataactagcagaacccgatccacttacattcgaataacgctgagaatccacaacgcttatggccagattctttccttcaatcttggcaaaatcaatgcctgcttgttttattatcacgaaagtactgaacaaccgagataggcacaataacaacaacagctttccatgatttggagactgatcggaatcgaatcagtgattttactggaagccgtttgattatgttttattatcacgaaagtactgaacaaccgagatagacacaataacaacaacaactttccatgatttggagactgatcagaatcgaaacagtgattttactttactggaagccgtttgattatttcctcttggatctcgaaaggcacgttgtctgacatcttgattttgattaccaactcaccatatcaatttctaggtagttaacaacgctaattagaaaaaaaaacgtgattcatatacacaacggtctctttgcttcgtccactagaactatttgtgtatgcattcttttggtagttaacaacgctaattagaaaaaaatctgtaaaattaatcatatcgattgctataacaggatggatgtgacacacacagatagataagctgtcacagttgtcaagactgtttgtcgggtttccaatgctcacacagggtgataaacaatgctgatacgaacacatgtgtttcgaaataggctttgttcttggtttaatgaagttacatgtaacatctacttatattcagatttaaacaatgcccgcctcaactgagataacggacatccatcaagctcaaaaagaaaacttgtgtttcccatagtcacaaaagattgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgcggtttgtatttcgcatagccaccgtttccgtagtaaaaaacggttgggtttgcacattcaatcttcaatacttcattaaaataccactagtagaagaacccgacacgtcttcatgttacggagaatgttctacaaattgaaggaaaaggaaaagatttgttgaaatcgaatttgacaactatgacagcttattaaaagtagcaatcacctccaatcatttcctggtcaaatcacacaccataaagtttaaagttatcgtgtgttgtagtttggaaagatgaccgaacaaagatctgaataacaaactgtggagaacttgtttgtgtcgggttcttctgctagttacttgatcaacggactgatcgttaacgttactctacttgtgctccgatttaagcggcgtgttccgatcaacgttgtttaaaggtgatgaagagtttagaaaccctagaatgatggtgcttttacgggaaaaaacgtgataattgaaaaacatcaatctatacggcctgtatacaattatacggcccgtatacatttggtaaacatcaaaaacctcccaaaataaatccattgagccgtatagtttttacaaatcgtatacacatgtatacggctcgtataactatatacgggccgtatatagtaaaaaaaacattctttacgcattgtcctattaaaaaacattctatacggaccgtatattttgtatacggtccgtataaactaaagatatgaaaataaaatatagggggtgtgggaataagaggagcgttattaaaatacacaaataattttcgtcatgactccaactacatgttttattgtgttcttgtttgtcgttattaatattttctttgacatagaaagtcgaaacgcatggtatccagctacgtacacatttattcaacttatatttattttagtcctcgtttgactttatgcctccactagctttctccatctgtttatcacgctgcaaataagtcaaacaattctccaccgtccgatcaaaacagtatctcattgactccactttcccaattggacctttataacccccttctacccctcatatctctcatcaatctctctttctctctcttagggtttcaacttccaaccttcttctacaacavariant HaWRK76 cDNA. This has a g in position 220 (underlined) andencodes a protein as shown in SEQ ID No. 12 has an A in the motif SRDAT instead of a T.SEQ ID NO: 11Atggcggttgatttcgtcggaattcaatctaccgatcatcttctaaaccgcatgttccagttattaagtcacgatttaaacgtttcgtcaacctacacgcacgcggtttctgctttcaaacgcaccggtcacgcacggttccgccgtggaccgtcgtctaccaccggagacactaacggaccttcaacttcttcacattcggaaggtaaatcacgagatgcgacttcgtttgtacaaaacgagtgtttttcaaacaaaccggtgacggagataacgacgacgacgacgtcaacgagctcgtcgtctgtagtatcgtcttccaccggtggaaacttagacggaagtgtttccaacggtaaacagttttcttcgttaggtatagtagctccggcgccgacgttctcgtctagaaaaccaccgttaccgtcgacacaccggaaaaggtgcggcgctgatcgtcctgttgcttccgtacacggatccggaagcggttgccattgttgttccaagagaaggaaaaccggatctaaacgtgaaattagaagagttccgattaccggatctaaaattacaagcatacctgctgatgattactcatggaaaaagtacggcgagaagaagatcgacggttcactttatccacgagtatattacaaatgtattaccggaaaaggatgtccggcgaggaagcgcgtggagttaagcgccgacgattcgaagatgcttattgttacttacgacggagaacaccgtcaccgtgaccgtcacgcgccggtacctatgagtttgaccggtgtgtatggtgagccaaagtgaavariant HaWRK76 protein. This has an A in the motif SRDAT instead of a T(see underlined A) SEQ ID NO: 12MAVDFVGIQSTDHLLNRMFQLLSHDLNVSSTYTHAVSAFKRTGHARFRRGPSSTTGDTNGPSTSSHSEGKSRDATSFVQNECFSNKPVTEITTTTTSTSSSSVVSSSTGGNLDGSVSNGKQFSSLGIVAPAPTFSSRKPPLPSTHRKRCGADRPVASVHGSGSGCHCCSKRRKTGSKREIRRVPITGSKITSIPADDYSWKKYGEKKIDGSLYPRVYYKCITGKGCPARKRVELSADDSKMLIVTYDGEHRHRDRHAPVPMSLIGVYGEPK

What is claimed is:
 1. An isolated polypeptide comprising a sequencehaving at least 90% sequence identity with the full-length amino acidsequence of SEQ ID NO: 2, the polypeptide sequence containing at leastone of: (a) proline as the amino acid corresponding to position 270 ofthe full-length amino acid sequence of SEQ ID NO: 2; (b) proline as theamino acid corresponding to position 87 of the full-length amino acidsequence of SEQ ID NO: 2; (c) proline as the amino acid corresponding toposition 260 of the full-length amino acid sequence of SEQ ID NO: 2; (d)serine as the amino acid corresponding to position 123 of thefull-length amino acid sequence of SEQ ID NO: 2; (e) leucine as theamino acid corresponding to position 14 of the full-length amino acidsequence of SEQ ID NO: 2; (f) leucine as the amino acid corresponding toposition 22 of the full-length amino acid sequence of SEQ ID NO: 2; and(g) serine as the amino acid corresponding to position 23 of thefull-length amino acid sequence of SEQ ID NO:
 2. 2. The isolatedpolypeptide of claim 1, wherein the polypeptide sequence containsproline as the amino acid corresponding to positions 270 and 87 of thefull-length amino acid sequence of SEQ ID NO:
 2. 3. The isolatedpolypeptide of claim 1, wherein the polypeptide sequence containsleucine and serine as the amino acids corresponding to positions 22 and23, respectively, of the full-length amino acid sequence of SEQ ID NO:2.
 4. The isolated polypeptide of claim 1, wherein the polypeptidecomprises a sequence having at least 95% sequence identity with thefull-length amino acid sequence of SEQ ID NO:
 2. 5. The isolatedpolypeptide of claim 1, wherein the polypeptide has the full-lengthamino acid sequence of SEQ ID NO: 2 or 12.